Fermentative production of ethanol from glucose, galactose and arabinose employing a recombinant yeast strain

ABSTRACT

The present invention relates to a process for the production of one or more fermentation product from a sugar composition, comprising the following steps: 
     a) fermentation of the sugar composition in the presence of a yeast belonging to the genera  Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces  or  Yarrowia ,: and 
     b) recovery of the fermentation product, 
     wherein the yeast comprises the genes araA, araB and araD and the sugar composition comprises glucose, galactose and arabinose.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a §371 National Stage Application ofPCT/EP2010/059618, filed Jul. 6, 2010, which claims priority to EuropeanApplication No. 09165229.7 filed Jul. 10, 2009.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates to mixed sugar fermentation, in particular thefermentation of a sugar composition comprising glucose, galactose andarabinose. The sugar composition may originate from ligno-cellulosicmaterial.

Description of Related Art

Most of the ethanol produced as alternative for fossil fuels iscurrently from fermentation of corn starch and sugar cane based sucrose.In order to reach the ambitious goals for producing renewable fuels, newtechnologies are being developed for converting non-food biomass intofermentation products such as ethanol. Saccharomyces cerevisiae is theorganism of choice in the ethanol industry, but it cannot utilizefive-carbon sugars contained in the hemicellulose component of biomassfeedstocks. Hemicellulose can make up to 20-30% of biomass, with xyloseand arabinose being the most abundant C5 sugars. Heterologous expressionof a xylose isomerase (XI) is an option for enabling yeast cells tometabolize and ferment xylose. Likewise, expression of bacterial genesaraA, araB, and araD in S. cerevisiae strains results in utilization andefficient alcoholic fermentation of arabinose. Galactose is a C6-sugarthat is also a sugar that is often present in lignocellulose, often inamounts (˜4% of total sugars) that are not to be neglected for economicreasons.

J. van den Brink et al, Microbiology (2009)155, 1340-1350 discloses thatglucose is the favoured carbon source for Saccharomyces cerevisiae andthat upon switching from glucose limited fermentation conditions togalactose-excess condition under anaerobic condition, galactose was notconsumed.

Sofar no process has been disclosed to convert galactose, into thefermentation product in the same process with glucose and one or more C5sugar. An object of the invention is therefore to provide a process toconvert galactose into the fermentation product in the same process withglucose and one or more C5 sugar.

SUMMARY

The present invention provides a process for the production of one ormore fermentation products from a sugar composition, comprising thefollowing steps:

-   -   a) fermentation of the sugar composition in the presence of a        yeast belonging to the genera Saccharomyces, Kluyveromyces,        Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera,        Schwanniomyces or Yarrowia,: and    -   b) recovery of the fermentation product,        -   wherein the yeast comprises the genes araA, araB and araD            and the sugar composition comprises glucose, galactose and            arabinose.

Advantageously the sugars glucose, galactose and arabinose are convertedinto fermentation product.

Preferably the mixed sugar cell is of the genus Saccharomyces morepreferably a Saccharomyces cerevisiae.

The invention further relates to the use of genes araA, araB and araD,to confer, through expression of those genes, on a glucose fermentingstrain the ability to anaerobically ferment galactose in the presence ofarabinose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets out a physical map of plasmid pPWT006.

FIG. 2 sets out a physical map of plasmid pPWT018.

FIG. 3 sets out a Southern blot autoradiogram. Chromosomal DNA ofwild-type strain CEN.PK113-7D (lane 1) and BIE104A2 (lane 2) wasdigested with both EcoRI and HindIII. The blot was hybridized with aspecific SIT2-probe.

FIG. 4 sets out physical maps of the wild-type SIT2-locus (panel a) andafter introduction of the ara-genes by integration of plasmid pPWT018,followed by intramolecular recombination leading to the loss of vectorand selectable marker sequences (panel b). The hybridization of theprobe is indicated.

FIG. 5 sets out a physical map of plasmid pPWT080, the sequence of whichis given in SEQ ID no. 4.

FIG. 6 sets out a physical map of the wild-type GRE3-locus (panel a) anda one copy integration of PWT080 in the GRE3-locus (panel b, showingwhere the primers bind and panel c, showing where the RKI1-probe binds).

FIG. 7 sets out a physical map of the GRE3-locus, where the codingregion of the GRE3-gene was replaced by the integration of the PPP-genesTAL1, TKL1, RKI1 and RPE1. Panel a shows the where the primers of SEQ ID5 and 6 bind, panel b shows where the RKI1-probe binds.

FIG. 8 sets out a growth curves under aerobic conditions of BIE104P1A2on different media. Strain BIE104A2P1 was pregrown on YNB 2% galactose.Growth curve was started on 2% galactose and 1% arabinose, followed byevent indicated in the graph by number (1) transfer to YNB with 2%arabinose as sole carbon source. After reaching an OD 600 more than 1,the culture was transferred to fresh medium with a starting OD 600 of0.2. Upon three transfers on pure arabinose medium the resulting strainwas designated BIE104P1A2c.

FIG. 9 sets out a growth curves under anaerobic conditions ofBIE104P1A2c on YNB 2% arabinose as sole carbon source. After reaching anOD 600 higher than 1, the culture was transferred to fresh medium with astarting OD 600 of 0.2. After several transfers the resulting strain wasnamed BIE104P1A2d (=BIE201).

FIG. 10 sets out sugar conversion and product formation of BIE104 onsynthetic corn fiber model medium. CO2 production was measuredconstantly. Growth was monitored by following optical density of theculture. Preculture was grown on 2% glucose.

FIG. 11 sets out sugar conversion and product formation of BIE104P1A2con synthetic corn fiber model medium. CO2 production was measuredconstantly. Growth was monitored by following optical density of theculture. Preculture was grown on 2% glucose.

FIG. 12 sets out sugar conversion and product formation of BIE201 onsynthetic corn fiber model medium. CO2 production was measuredconstantly. Growth was monitored by following optical density of theculture. Preculture was grown on 2% glucose.

FIG. 13 sets out sugar conversion and product formation of BIE104A2 onsynthetic corn fiber model medium. CO2 production was measuredconstantly. Growth was monitored by following optical density of theculture. Preculture was grown on 2% glucose.

FIG. 14 sets out sugar conversion and product formation of BIE105A2 onsynthetic corn fiber model medium. CO2 production was measuredconstantly. Growth was monitored by following optical density of theculture. Preculture was grown on 2% glucose.

FIG. 15 sets out a physical map of plasmid pPWT007

FIG. 16 sets out a physical map of plasmid pPWT042

FIG. 17 sets out a physical map of the wild-type SIT4-locus (panel a)and a one copy integration of PWT080 in the SIT4-locus (panel b, showingwhere the primers bind).

FIG. 18 sets out a graphic representation of growth curves of strainBIE104A2P1Y9 on different media. Panel a: strain BIE104A2P1Y9 grown onglucose, followed by events indicated in the graph by numbers (1)transfer to 1% arabinose+1% xylose and (2) transfer to 2% xylose+0.2%arabinose. Panel b: strain BIE104A2P1Y9 grown on galactose, followed by(1) transfer to 1% arabinose+1% xylose and (2) transfer to 2%xylose+0.2% arabinose.

FIG. 19 sets out growth on Verduyn-medium supplemented with 2% xylose ofstrain BIE104A2P1Y9. Two independent colonies were tested. Afterreaching on OD 600 of 2, strains were transferred to fresh medium andimmediately started to grow again on xylose.

FIG. 20 sets out a physical map of plasmid pGBS416ARAABD.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

SEQ ID NO: 1 sets out the wild-type xylose isomerase sequence fromBacteroides uniformis ATCC 8492. Genbank accession no. AAYH02000036.

SEQ ID NO: 2 sets out a codon optimized sequence derived from SEQ ID NO:1.

SEQ ID NO: 3 sets out the amino acid sequence of xylose isomerase fromBacteroides uniformis ATCC 8492.

SEQ ID NO: 4 sets out the sequence of plasmid pPWT080.

SEQ ID NO: 5 sets out the sequence of forward primer.

SEQ ID NO: 6 sets out the sequence of reverse primer.

SEQ ID NO: 7 sets out the sequence of the forward multifunctional primerfor diagnostic PCR.

SEQ ID NO: 8 sets out the sequence of reverse multifunctional primer fordiagnostic PCR.

SEQ ID NO: 9 sets out the sequence of forward primer RKI1-probe.

SEQ ID NO: 10 sets out the sequence of reverse primer RKI1-probe.

SEQ ID NO: 11 sets out the sequence of forward primer kanMX-cassette.

SEQ ID NO: 12 sets out the sequence of reverse primer kanMX-cassette.

SEQ ID NO: 13 sets out the sequence of forward primer.

SEQ ID NO: 14 sets out the sequence of reverse primer.

SEQ ID NO: 15 sets out the sequence of forward multifunctional primerfor diagnostic PCR.

SEQ ID NO: 16 sets out the sequence of reverse multifunctional primerfor diagnostic PCR.

SEQ ID NO: 17 sets out the sequence of sequence of plasmid pPWT018

SEQ ID NO: 18 sets out the sequence of forward primer integrationpPWT018.

SEQ ID NO: 19 sets out the sequence of reverse primer integrationpPWT018.

SEQ ID NO: 20 sets out the sequence of forward primer SIT2-probe.

SEQ ID NO: 21 sets out the sequence of reverse primer SIT2-probe.

SEQ ID NO: 22 sets out the sequence of forward primer to amplify araABDexpression cassette.

SEQ ID NO: 23 sets out the sequence of reverse primer to amplify araABDexpression cassette.

Throughout the present specification and the accompanying claims, thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to one or at least one) of the grammatical object of thearticle. By way of example, “an element” may mean one element or morethan one element.

The various embodiments of the invention described herein may becross-combined.

The Sugar Composition

The sugar composition according to the invention comprises glucose,arabinose and galactose. In the process of the invention, advantageouslythe sugars glucose, galactose and arabinose are converted intofermentation product.

Any sugar composition may be used in the invention that suffices thosecriteria. In a preferred embodiment, the sugar composition is ahydrolysate of one or more lignocellulosic material. Lignocelluloseherein includes hemicellulose and hemicellulose parts of biomass. Alsolignocellulose includes lignocellulosic fractions of biomass. Suitablelignocellulosic materials may be found in the following list: orchardprimings, chaparral, mill waste, urban wood waste, municipal waste,logging waste, forest thinnings, short-rotation woody crops, industrialwaste, wheat straw, oat straw, rice straw, barley straw, rye straw, flaxstraw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls,sugar cane, corn stover, corn stalks, corn cobs, corn husks, switchgrass, miscanthus, sweet sorghum, canola stems, soybean stems, prairiegrass, gamagrass, foxtail; sugar beet pulp, citrus fruit pulp, seedhulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, trees,softwood, hardwood, poplar, pine, shrubs, grasses, wheat, wheat straw,sugar cane bagasse, corn, corn husks, corn hobs, corn kernel, fiber fromkernels, products and by-products from wet or dry milling of grains,municipal solid waste, waste paper, yard waste, herbaceous material,agricultural residues, forestry residues, municipal solid waste, wastepaper, pulp, paper mill residues, branches, bushes, canes, corn, cornhusks, an energy crop, forest, a fruit, a flower, a grain, a grass, aherbaceous crop, a leaf, bark, a needle, a log, a root, a sapling, ashrub, switch grass, a tree, a vegetable, fruit peel, a vine, sugar beetpulp, wheat midlings, oat hulls, hard or soft wood, organic wastematerial generated from an agricultural process, forestry wood waste, ora combination of any two or more thereof.

An overview of some suitable sugar compositions derived fromlignocellulose and the sugar composition of their hydrolysates is givenin table 1. The listed lignocelluloses include: corn cobs, corn fiber,rice hulls, melon shells, sugar beet pulp, wheat straw, sugar canebagasse, wood, grass and olive pressings.

TABLE 1 Overview of sugar compositions from lignocellulosic materials.Lignocellulosic material Gal Xyl Ara Man Glu Rham Sum %. Gal. Lit. Corncob a 10 286 36 227 11 570 1.7 (1) Corn cob b 131 228 160 144 663 19.8(1) Rice hulls a 9 122 24 18 234 10 417 2.2 (1) Rice hulls b 8 120 28209 12 378 2.2 (1) Melon Shells 6 120 11 208 16 361 1.7 (1) Sugar beetpulp 51 17 209 11 211 24 523 9.8 (2) Whea straw Idaho 15 249 36 396 6962.2 (3) Corn fiber 36 176 113 372 697 5.2 (4) Cane Bagasse 14 180 24 5391 614 2.3 (5) Corn stover 19 209 29 370 626 (6) Athel (wood) 5 118 7 3493 625 0.7 (7) Eucalyptus (wood) 22 105 8 3 445 583 3.8 (7) CWR (grass)8 165 33 340 546 1.4 (7) JTW (grass) 7 169 28 311 515 1.3 (7) MSW 4 24 520 440 493 0.9 (7) Reed Canary Grass Veg 16 117 30 6 209 1 379 4.2 (8)Reed Canary Grass Seed 13 163 28 6 265 1 476 2.7 (9) Olive pressingresidu 15 111 24 8 329 487 3.1 (9) Avg 3.8 Gal = galactose, Xyl =xylose, Ara = arabinose, Man = mannose, Glu = glutamate, Rham =rhamnose. The percentage galactose (% Gal) and literature source isgiven.

It is clear from table 1 that in these lignocelluloses a considerableamount of sugar (on average 3.8%) is galactose. The conversion ofgalactose to fermentation product is thus of great economic importance.

The Mixed Sugar Cell

The mixed sugar cell comprising the genes araA, araB and araD as definedhereafter. It is able to ferment glucose, arabinose and galactose. Inone embodiment of the invention the mixed sugar cell is able to fermentone or more additional sugar, preferably C5 and/or C6 sugar. In anembodiment of the invention the mixed sugar cell comprises one or moreof: a xylA-gene and/or XKS1-gene, to allow the mixed sugar cell toferment xylose; deletion of the aldose reductase (GRE3) gene;overexpression of PPP-genes TAL1, TKL1, RPE1 and RKI1 to allow theincrease of the flux through the pentose phosphate pass-way in the cell.

In one embodiment of the invention the mixed sugar cell is able toferment one or more additional sugar, preferably C5 and/or C6 sugars. Inan embodiment of the invention the mixed sugar cell comprises one ormore of: a xylA-gene, XYL1 gene and XYL2 gene and/or XKS1-gene, to allowthe mixed sugar cell to ferment xylose; deletion of the aldose reductase(GRE3) gene; overexpression of PPP-genes TAL1, TKL1, RPE1 and RKI1 toallow the increase of the flux through the pentose phosphate pass-way inthe cell.

In an embodiment, the mixed sugar cell is an industrial cell, morepreferably an industrial yeast. An industrial cell and industrial yeastcell may be defined as follows. The living environments of (yeast) cellsin industrial processes are significantly different from that in thelaboratory. Industrial yeast cells must be able to perform well undermultiple environmental conditions which may vary during the process.Such variations include change in nutrient sources, pH, ethanolconcentration, temperature, oxygen concentration, etc., which togetherhave potential impact on the cellular growth and ethanol production ofSaccharomyces cerevisiae. Under adverse industrial conditions, theenvironmental tolerant strains should allow robust growth andproduction. Industrial yeast strains are generally more robust towardsthese changes in environmental conditions which may occur in theapplications they are used, such as in the baking industry, brewingindustry, wine making and the ethanol industry. In one embodiment, theindustrial mixed sugar cell is constructed on the basis of an industrialhost cell, wherein the construction is conducted as describedhereinafter. Examples of industrial yeast (S. cerevisiae) are EthanolRed® (Fermentis) Fermiol® (DSM) and Thermosacc® (Lallemand).

In an embodiment the mixed sugar cell is inhibitor tolerant. Inhibitortolerance is resistance to inhibiting compounds. The presence and levelof inhibitory compounds in lignocellulose may vary widely with variationof feedstock, pretreatment method hydrolysis process. Examples ofcategories of inhibitors are carboxylic acids, furans and/or phenoliccompounds. Examples of carboxylic acids are lactic acid, acetic acid orformic acid. Examples of furans are furfural and hydroxy-methylfurfural.Examples or phenolic compounds are vannilin, syringic acid, ferulic acidand coumaric acid. The typical amounts of inhibitors are for carboxylicacids: several grams per liter, up to 20 grams per liter or more,depending on the feedstock, the pretreatment and the hydrolysisconditions. For furans: several hundreds of milligrams per liter up toseveral grams per liter, depending on the feedstock, the pretreatmentand the hydrolysis conditions.

For phenolics: several tens of milligrams per liter, up to a gram perliter, depending on the feedstock, the pretreatment and the hydrolysisconditions.

The mixed sugar strains according to the invention are inhibitortolerant, i.e. they can withstand common inhibitors at the level thatthey typically have with common pretreatment and hydrolysis conditions,so that the mixed sugar strains can find broad application, i.e. it hashigh applicability for different feedstock, different pretreatmentmethods and different hydrolysis conditions.

In one embodiment, the industrial mixed sugar cell is constructed on thebasis of an inhibitor tolerant host cell, wherein the construction isconducted as described hereinafter. Inhibitor tolerant host cells may beselected by screening strains for growth on inhibitors containingmaterials, such as illustrated in Kadar et al, Appl. Biochem.Biotechnol. (2007), Vol. 136-140, 847-858, wherein an inhibitor tolerantS. cerevisiae strain ATCC 26602 was selected.

In an embodiment, the mixed sugar cell is marker-free. As used herein,the term “marker” refers to a gene encoding a trait or a phenotype whichpermits the selection of, or the screening for, a host cell containingthe marker. Marker-free means that markers are essentially absent in themixed sugar cell. Being marker-free is particularly advantageous whenantibiotic markers have been used in construction of the mixed sugarcell and are removed thereafter. Removal of markers may be done usingany suitable prior art technique, e.g intramolecular recombination. Asuitable method of marker removal is illustrated in the examples.

A mixed sugar cell may be able to convert plant biomass, celluloses,hemicelluloses, pectins, rhamnose, galactose, frucose, maltose,maltodextrines, ribose, ribulose, or starch, starch derivatives,sucrose, lactose and glycerol, for example into fermentable sugars.Accordingly, a mixed sugar cell may express one or more enzymes such asa cellulase (an endocellulase or an exocellulase), a hemicellulase (anendo- or exo-xylanase or arabinase) necessary for the conversion ofcellulose into glucose monomers and hemicellulose into xylose andarabinose monomers, a pectinase able to convert pectins into glucuronicacid and galacturonic acid or an amylase to convert starch into glucosemonomers.

The mixed sugar cell further may comprise those enzymatic activitiesrequired for conversion of pyruvate to a desired fermentation product,such as ethanol, butanol, lactic acid, 3-hydroxy-propionic acid, acrylicacid, acetic acid, succinic acid, citric acid, fumaric acid, malic acid,itaconic acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, aβ-lactam antibiotic or a cephalosporin.

In an embodiment, the mixed sugar cell a cell that is naturally capableof alcoholic fermentation, preferably, anaerobic alcoholic fermentation.A mixed sugar cell preferably has a high tolerance to ethanol, a hightolerance to low pH (i.e. capable of growth at a pH lower than about 5,about 4, about 3, or about 2.5) and towards organic and/or a hightolerance to elevated temperatures.

Any of the above characteristics or activities of a mixed sugar cell maybe naturally present in the cell or may be introduced or modified bygenetic modification.

Construction of the Mixed Sugar Strain

The genes may be introduced in the mixed sugar cell by introduction intoa host cell:

-   -   a) a cluster consisting of PPP-genes TAL1, TKL1, RPE1 and RKI1,        under control of strong promoters,    -   b) a cluster consisting of a xylA-gene and the XKS1-gene both        under control of constitutive promoters,    -   c) a cluster consisting of the genes araA, araB and araD and/or        a cluster of xylA-gene and/or the XKS1-gene;        and    -   d) deletion of an aldose reductase gene        and adaptive evolution to produce the mixed sugar cell. The        above cell may be constructed using recombinant expression        techniques.

Recombinant Expression

The cell of the invention is a recombinant cell. That is to say, a cellof the invention comprises, or is transformed with or is geneticallymodified with a nucleotide sequence that does not naturally occur in thecell in question.

Techniques for the recombinant expression of enzymes in a cell, as wellas for the additional genetic modifications of a cell of the inventionare well known to those skilled in the art. Typically such techniquesinvolve transformation of a cell with nucleic acid construct comprisingthe relevant sequence. Such methods are, for example, known fromstandard handbooks, such as Sambrook and Russel (2001) “MolecularCloning: A Laboratory Manual (3rd edition), Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al.,eds., “Current protocols in molecular biology”, Green Publishing andWiley Interscience, New York (1987). Methods for transformation andgenetic modification of fungal host cells are known from e.g. EP-A-0635574, WO 98/46772, WO 99/60102, WO 00/37671, WO90/14423, EP-A-0481008,EP-A-0635574 and U.S. Pat. No. 6,265,186.

Typically, the nucleic acid construct may be a plasmid, for instance alow copy plasmid or a high copy plasmid. The cell according to thepresent invention may comprise a single or multiple copies of thenucleotide sequence encoding a enzyme, for instance by multiple copiesof a nucleotide construct or by use of construct which has multiplecopies of the enzyme sequence.

The nucleic acid construct may be maintained episomally and thuscomprise a sequence for autonomous replication, such as an autosomalreplication sequence sequence. A suitable episomal nucleic acidconstruct may e.g. be based on the yeast 2μ or pKD1 plasmids (Gleer etal., 1991, Biotechnology 9: 968-975), or the AMA plasmids (Fierro etal., 1995, Curr Genet. 29:482-489). Alternatively, each nucleic acidconstruct may be integrated in one or more copies into the genome of thecell. Integration into the cell's genome may occur at random bynon-homologous recombination but preferably, the nucleic acid constructmay be integrated into the cell's genome by homologous recombination asis well known in the art (see e.g. WO90/14423, EP-A-0481008, EP-A-0635574 and U.S. Pat. No. 6,265,186).

Most episomal or 2μ plasmids are relatively unstable, being lost inapproximately 10⁻² or more cells after each generation. Even underconditions of selective growth, only 60% to 95% of the cells retain theepisomal plasmid. The copy number of most episomal plasmids ranges from10-40 per cell of cir⁺ hosts. However, the plasmids are not equallydistributed among the cells, and there is a high variance in the copynumber per cell in populations. Strains transformed with integrativeplasmids are extremely stable, even in the absence of selectivepressure. However, plasmid loss can occur at approximately 10⁻³ to 10⁻⁴frequencies by homologous recombination between tandemly repeated DNA,leading to looping out of the vector sequence. Preferably, the vectordesign in the case of stable integration is thus, that upon loss of theselection marker genes (which also occurs by intramolecular, homologousrecombination) that looping out of the integrated construct is no longerpossible. Preferably the genes are thus stably integrated. Stableintegration is herein defined as integration into the genome, whereinlooping out of the integrated construct is no longer possible.Preferably selection markers are absent. Typically, the enzyme encodingsequence will be operably linked to one or more nucleic acid sequences,capable of providing for or aiding the transcription and/or translationof the enzyme sequence.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. For instance, a promoter or enhancer isoperably linked to a coding sequence the said promoter or enhanceraffects the transcription of the coding sequence.

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequencesknown to one of skilled in the art. A “constitutive” promoter is apromoter that is active under most environmental and developmentalconditions. An “inducible” promoter is a promoter that is active underenvironmental or developmental regulation.

The promoter that could be used to achieve the expression of anucleotide sequence coding for an enzyme according to the presentinvention, may be not native to the nucleotide sequence coding for theenzyme to be expressed, i.e. a promoter that is heterologous to thenucleotide sequence (coding sequence) to which it is operably linked.The promoter may, however, be homologous, i.e. endogenous, to the hostcell.

Promotors are widely available and known to the skilled person. Suitableexamples of such promoters include e.g. promoters from glycolytic genes,such as the phosphofructokinase (PFK), triose phosphate isomerase (TPI),glyceraldehyde-3-phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvatekinase (PYK), phosphoglycerate kinase (PGK) promoters from yeasts orfilamentous fungi; more details about such promoters from yeast may befound in (WO 93/03159). Other useful promoters are ribosomal proteinencoding gene promoters, the lactase gene promoter (LAC4), alcoholdehydrogenase promoters (ADHI, ADH4, and the like), and the enolasepromoter (ENO). Other promoters, both constitutive and inducible, andenhancers or upstream activating sequences will be known to those ofskill in the art. The promoters used in the host cells of the inventionmay be modified, if desired, to affect their control characteristics.Suitable promoters in this context include both constitutive andinducible natural promoters as well as engineered promoters, which arewell known to the person skilled in the art. Suitable promoters ineukaryotic host cells may be GAL7, GAL10, or GAL1, CYC1, HIS3, ADH1,PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO1, TPI1, and AOX1. Othersuitable promoters include PDC1, GPD1, PGK1, TEF1, and TDH3.

In a cell of the invention, the 3′-end of the nucleotide acid sequenceencoding enzyme preferably is operably linked to a transcriptionterminator sequence. Preferably the terminator sequence is operable in ahost cell of choice, such as e.g. the yeast species of choice. In anycase the choice of the terminator is not critical; it may e.g. be fromany yeast gene, although terminators may sometimes work if from anon-yeast, eukaryotic, gene. Usually a nucleotide sequence encoding theenzyme comprises a terminator. Preferably, such terminators are combinedwith mutations that prevent nonsense mediated mRNA decay in the hostcell of the invention (see for example: Shirley et al., 2002, Genetics161:1465-1482).

The transcription termination sequence further preferably comprises apolyadenylation signal.

Optionally, a selectable marker may be present in a nucleic acidconstruct suitable for use in the invention. As used herein, the term“marker” refers to a gene encoding a trait or a phenotype which permitsthe selection of, or the screening for, a host cell containing themarker. The marker gene may be an antibiotic resistance gene whereby theappropriate antibiotic can be used to select for transformed cells fromamong cells that are not transformed. Examples of suitable antibioticresistance markers include e.g. dihydrofolate reductase,hygromycin-B-phosphotransferase, 3′-O-phosphotransferase II (kanamycin,neomycin and G418 resistance). Antibiotic resistance markers may be mostconvenient for the transformation of polyploid host cells, Alsonon-antibiotic resistance markers may be used, such as auxotrophicmarkers (URA3, TRPI, LEU2) or the S. pombe TPI gene (described byRussell P R, 1985, Gene 40: 125-130). In a preferred embodiment the hostcells transformed with the nucleic acid constructs are marker gene free.Methods for constructing recombinant marker gene free microbial hostcells are disclosed in EP-A-0 635 574 and are based on the use ofbidirectional markers such as the A. nidulans amdS (acetamidase) gene orthe yeast URA3 and LYS2 genes. Alternatively, a screenable marker suchas Green Fluorescent Protein, lacL, luciferase, chloramphenicolacetyltransferase, beta-glucuronidase may be incorporated into thenucleic acid constructs of the invention allowing to screen fortransformed cells.

Optional further elements that may be present in the nucleic acidconstructs suitable for use in the invention include, but are notlimited to, one or more leader sequences, enhancers, integrationfactors, and/or reporter genes, intron sequences, centromers, telomersand/or matrix attachment (MAR) sequences. The nucleic acid constructs ofthe invention may further comprise a sequence for autonomousreplication, such as an ARS sequence.

The recombination process may thus be executed with known recombinationtechniques. Various means are known to those skilled in the art forexpression and overexpression of enzymes in a cell of the invention. Inparticular, an enzyme may be overexpressed by increasing the copy numberof the gene coding for the enzyme in the host cell, e.g. by integratingadditional copies of the gene in the host cell's genome, by expressingthe gene from an episomal multicopy expression vector or by introducinga episomal expression vector that comprises multiple copies of the gene.

Alternatively, overexpression of enzymes in the host cells of theinvention may be achieved by using a promoter that is not native to thesequence coding for the enzyme to be overexpressed, i.e. a promoter thatis heterologous to the coding sequence to which it is operably linked.Although the promoter preferably is heterologous to the coding sequenceto which it is operably linked, it is also preferred that the promoteris homologous, i.e. endogenous to the host cell. Preferably theheterologous promoter is capable of producing a higher steady statelevel of the transcript comprising the coding sequence (or is capable ofproducing more transcript molecules, i.e. mRNA molecules, per unit oftime) than is the promoter that is native to the coding sequence.Suitable promoters in this context include both constitutive andinducible natural promoters as well as engineered promoters.

In an embodiment, the mixed sugar cell is markerfree, which means thatno auxotrophic or dominant markers, in particular antibiotic resistancemarkers, are present in the genome or extra-chromosomally.

The coding sequence used for overexpression of the enzymes mentionedabove may preferably be homologous to the host cell of the invention.However, coding sequences that are heterologous to the host cell of theinvention may be used.

Overexpression of an enzyme, when referring to the production of theenzyme in a genetically modified cell, means that the enzyme is producedat a higher level of specific enzymatic activity as compared to theunmodified host cell under identical conditions. Usually this means thatthe enzymatically active protein (or proteins in case of multi-subunitenzymes) is produced in greater amounts, or rather at a higher steadystate level as compared to the unmodified host cell under identicalconditions. Similarly this usually means that the mRNA coding for theenzymatically active protein is produced in greater amounts, or againrather at a higher steady state level as compared to the unmodified hostcell under identical conditions. Preferably in a host cell of theinvention, an enzyme to be overexpressed is overexpressed by at least afactor of about 1.1, about 1.2, about 1.5, about 2, about 5, about 10 orabout 20 as compared to a strain which is genetically identical exceptfor the genetic modification causing the overexpression. It is to beunderstood that these levels of overexpression may apply to the steadystate level of the enzyme's activity, the steady state level of theenzyme's protein as well as to the steady state level of the transcriptcoding for the enzyme.

Adaptation

Adaptation is the evolutionary process whereby a population becomesbetter suited (adapted) to its habitat or habitats. This process takesplace over several to many generations, and is one of the basicphenomena of biology.

The term adaptation may also refer to a feature which is especiallyimportant for an organism's survival. Such adaptations are produced in avariable population by the better suited forms reproducing moresuccessfully, by natural selection.

Changes in environmental conditions alter the outcome of naturalselection, affecting the selective benefits of subsequent adaptationsthat improve an organism's fitness under the new conditions. In the caseof an extreme environmental change, the appearance and fixation ofbeneficial adaptations can be essential for survival. A large number ofdifferent factors, such as e.g. nutrient availability, temperature, theavailability of oxygen, etcetera, can drive adaptive evolution.

Fitness

There is a clear relationship between adaptedness (the degree to whichan organism is able to live and reproduce in a given set of habitats)and fitness. Fitness is an estimate and a predictor of the rate ofnatural selection. By the application of natural selection, the relativefrequencies of alternative phenotypes will vary in time, if they areheritable.

Genetic Changes

When natural selection acts on the genetic variability of thepopulation, genetic changes are the underlying mechanism. By this means,the population adapts genetically to its circumstances. Genetic changesmay result in visible structures, or may adjust the physiologicalactivity of the organism in a way that suits the changed habitat.

The Adaptive Evolution

The mixed sugar cells are in their preparation subjected to adaptiveevolution. A cell of the invention may be adapted to sugar utilisationby selection of mutants, either spontaneous or induced (e.g. byradiation or chemicals), for growth on the desired sugar, preferably assole carbon source, and more preferably under anaerobic conditions.Selection of mutants may be performed by techniques including serialtransfer of cultures as e.g. described by Kuyper et al. (2004, FEMSYeast Res. 4: 655-664) or by cultivation under selective pressure in achemostat culture. E.g. in a preferred host cell of the invention atleast one of the genetic modifications described above, includingmodifications obtained by selection of mutants, confer to the host cellthe ability to grow on the xylose as carbon source, preferably as solecarbon source, and preferably under anaerobic conditions. Preferably thecell produce essentially no xylitol, e.g. the xylitol produced is belowthe detection limit or e.g. less than about 5, about 2, about 1, about0.5, or about 0.3% of the carbon consumed on a molar basis.

Adaptive evolution is also described e.g. in Wisselink H. W. et al,Applied and Environmental Microbiology August 2007, p. 4881-4891

In one embodiment of adaptive evolution a regimen consisting of repeatedbatch cultivation with repeated cycles of consecutive growth indifferent media is applied, e.g. three media with different compositions(glucose, xylose, and arabinose; xylose and arabinose. See Wisselink etal. (2009) Applied and Environmental Microbiology, February 2009, p.907-914.

Yeast Transformation and Genetic Stability

Genetic engineering, i.e. transformation of yeast cells with recombinantDNA, became feasible for the first time in 1978 [Beggs, 1978; Hinnen etal., 1978]. Recombinant DNA technology in yeast has established itselfsince then. A multitude of different vector constructs are available.Generally, these plasmid vectors, called shuttle vectors, containgenetic material derived from E. coli vectors consisting of an origin ofreplication and a selectable marker (often the βlactamase gene, ampR),which enable them to be propagated in E. coli prior to transformationinto yeast cells. Additionally, the shuttle vectors contain a selectablemarker for selection in yeast. Markers can be genes encoding enzymes forthe synthesis of a particular amino acid or nucleotide, so that cellscarrying the corresponding genomic deletion (or mutation) arecomplemented for auxotrophy or autotrophy. Alternatively, these vectorscontain heterologous dominant resistance markers, which providesrecombinant yeast cells (i.e. the cells that have taken up the DNA andexpress the marker gene) resistance towards certain antibiotics, likeg418 (Geneticin), hygromycinB or phleomycin. In addition, these vectorsmay contain a sequence of (combined) restriction sites (multiple cloningsite or MCS) which will allow to clone foreign DNA into these sites,although alternative methods exist as well.

Traditionally, four types of shuttle vectors can be distinguished by theabsence or presence of additional genetic elements:

-   -   Integrative plasmids (YIp) which by homologous recombination are        integrated into the host genome at the locus of the marker or        another gene, when this is opened by restriction and the        linearized DNA is used for transformation of the yeast cells.        This generally results in the presence of one copy of the        foreign DNA inserted at this particular site in the genome.    -   Episomal plasmids (YEp) which carry part of the 2μ plasmid DNA        sequence necessary for autonomous replication in yeast cells.        Multiple copies of the transformed plasmid are propagated in the        yeast cell and maintained as episomes.    -   Autonomously replicating plasmids (YRp) which carry a yeast        origin of replication (ARS, autonomously replicated sequence)        that allows the transformed plasmids to be propagated several        hundred-fold.    -   CEN plasmids (YCp) which carry in addition to an ARS sequence a        centromeric sequence (derived from one of the nuclear        chromosomes) which normally guarantees stable mitotic        segregation and usually reduces the copy number of        self-replicated plasmid to just one.

These plasmids are being introduced into the yeast cells bytransformation. Transformation of yeast cells may be achieved by severaldifferent techniques, such as permeabilization of cells with lithiumacetate (Ito et al, 1983) and electroporation methods.

In commercial application of recombinant microorganisms, plasmidinstability is the most important problem. Instability is the tendencyof the transformed cells to lose their engineered properties because ofchanges to, or loss of, plasmids. This issue is discussed in detail byZhang et al (Plasmid stability in recombinant Saccharomyces cerevisiae.Biotechnology Advances, Vol. 14, No. 4, pp. 401-435, 1996). Strainstransformed with integrative plasmids are extremely stable, even in theabsence of selective pressure (Sherman, F.dbb.urmc.rochester.edu/labs/sherman f/yeast/9.html and referencestherein).

The heterologous DNA is usually introduced into the organism in the formof extra-chromosomal plasmids (YEp, YCp and YRp). Unfortunately, it hasbeen found with both bacteria and yeasts that the new characteristicsmay not be retained, especially if the selection pressure is not appliedcontinuously. This is due to the segregational instability of the hybridplasmid when recombinant cells grow for a long period of time. Thisleads to population heterogeneity and clonal variability, and eventuallyto a cell population in which the majority of the cells has lost theproperties that were introduced by transformation. If vectors withauxotrophic markers are being used, cultivation in rich media oftenleads to rapid loss of the vector, since the vector is only retained inminimal media. The alternative, the use of dominant antibioticresistance markers, is often not compatible with production processes.The use of antibiotics may not be desired from a registration point ofview (the possibility that trace amounts of the antibiotic end up in theend product) or for economic reasons (costs of the use of antibiotics atindustrial scale).

Loss of vectors leads to problems in large scale production situations.Alternative methods for introduction of DNA do exist for yeasts, such asthe use of integrating plasmids (YIp). The DNA is integrated into thehost genome by recombination, resulting in high stability. (Caunt, P.Stability of recombinant plasmids in yeast. Journal of Biotechnology 9(1988) 173-192). We have found that an integration method using the hosttransposons are a good alternative.

Transposons

In an embodiment of the invention, the cell may comprise more than onecopy of desired gene(s). For instance, two or more xylose isomerase geneor xylose reductase gene and xylitol dehydrogenase may be integratedinto the mixed sugar cell genome. This may be executed in any way knownin the art that leads to introduction of the genes. In a preferredembodiment, this may be accomplished using a vector with partshomologous to repeated sequences (transposons), of the host cell. Whenthe host cell is a yeast cell, suitable repeated sequences are the longterminal repeats (LTR) of the Ty element, known as delta sequence.

Ty elements fall into two rather similar subfamilies called Ty1 and Ty2.These elements are about 6 kilobases (kb) in length and are bounded bylong terminal repeats (LTR), sequences of about 335 base pairs (Boeke JD et al, The Saccharomyces cerevisiae Genome Contains Functional andNonfunctional Copies of Transposon Ty1. Molecular and Cellular Biology,April 1988, p. 1432-1442 Vol. 8, No. 4). In the fully sequenced S.cerevisiae strain, S288c, the most abundant transposons are Ty1 (31copies) and Ty2 (13 copies) (Gabriel A, Dapprich J, Kunkel M, Gresham D,Pratt S C, et al. (2006) Global mapping of transposon location. PLoSGenet. 2(12): e212.doi:10.1371/journal.pgen.0020212). These transposonsconsist of two overlapping open reading frames (ORFs), each of whichencode several proteins. The coding regions are flanked by theaforementioned, nearly identical LTRs. Other, but less abundant and moredistinct Ty elements in S. cereviaise comprise Ty3, Ty4 and Ty5. Foreach family of full-length Ty elements there are an order of magnitudemore solo LTR elements dispersed through the genome. These are thoughtto arise by LTR-LTR recombination of full-length elements, with loopingout of the internal protein encoding regions.

The retrotransposition mechanism of the Ty retrotransposon has beenexploited to integrate multiple copies throughout the genome (Boeke etal., 1988; Jacobs et al., 1988). The long terminal repeats (LTR) of theTy element, known as delta sequences, are also good targets forintegration by homologous recombination as they exist in about 150-200copies that are either Ty associated or solo sites (Boeke, 1989;Kingsman and Kingsman, 1988). (Parekh R. N. (1996). An IntegratingVector for Tunable, High Copy, Stable Integration into the Dispersed TyDELTA Sites of Saccharomyces cerevisiae. Biotechnol. Prog. 1996, 12,16-21).

The Host Cell

The host cell may be any host cell suitable for production of a usefulproduct. A cell of the invention may be any suitable cell, such as aprokaryotic cell, such as a bacterium, or a eukaryotic cell. Typically,the cell will be a eukaryotic cell, for example a yeast or a filamentousfungus.

Yeasts are herein defined as eukaryotic microorganisms and include allspecies of the subdivision Eumycotina (Alexopoulos, C. J., 1962, In:Introductory Mycology, John Wiley & Sons, Inc., New York) thatpredominantly grow in unicellular form.

Yeasts may either grow by budding of a unicellular thallus or may growby fission of the organism. A preferred yeast as a cell of the inventionmay belong to the genera Saccharomyces, Kluyveromyces, Candida, Pichia,Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces or Yarrowia.Preferably the yeast is one capable of anaerobic or oxygen limitedfermentation, more preferably one capable of anaerobic alcoholicfermentation.

Filamentous fungi are herein defined as eukaryotic microorganisms thatinclude all filamentous forms of the subdivision Eumycotina. These fungiare characterized by a vegetative mycelium composed of chitin,cellulose, and other complex polysaccharides. The filamentous fungi ofthe suitable for use as a cell of the present invention aremorphologically, physiologically, and genetically distinct from yeasts.Filamentous fungal cells may be advantageously used since most fungi donot require sterile conditions for propagation and are insensitive tobacteriophage infections. Vegetative growth by filamentous fungi is byhyphal elongation and carbon catabolism of most filamentous fungi isobligately aerobic. Preferred filamentous fungi as a host cell of theinvention may belong to the genus Aspergillus, Trichoderma, Humicola,Acremoniurra, Fusarium or Penicillium. More preferably, the filamentousfungal cell may be a Aspergillus niger, Aspergillus oryzae, aPenicillium chrysogenum, or Rhizopus oryzae cell.

In one embodiment the host cell may be yeast.

Preferably the host is an industrial host, more preferably an industrialyeast. An industrial host and industrial yeast cell may be defined asfollows. The living environments of yeast cells in industrial processesare significantly different from that in the laboratory. Industrialyeast cells must be able to perform well under multiple environmentalconditions which may vary during the process. Such variations includechange in nutrient sources, pH, ethanol concentration, temperature,oxygen concentration, etc., which together have potential impact on thecellular growth and ethanol production of Saccharomyces cerevisiae.Under adverse industrial conditions, the environmental tolerant strainsshould allow robust growth and production. Industrial yeast strains aregenerally more robust towards these changes in environmental conditionswhich may occur in the applications they are used, such as in the bakingindustry, brewing industry, wine making and the ethanol industry.Examples of industrial yeast (S. cerevisiae) are Ethanol Red®(Fermentis), Fermiol® (DSM) and Thermosacc® (Lallemand).

In an embodiment the host is inhibitor tolerant. Inhibitor tolerant hostcells may be selected by screening strains for growth on inhibitorscontaining materials, such as illustrated in Kadar et al, Appl. Biochem.Biotechnol. (2007), Vol. 136-140, 847-858, wherein an inhibitor tolerantS. cerevisiae strain ATCC 26602 was selected.

Preferably the host cell is industrial and inhibitor tolerant.

AraA, AraB and AraD Genes

A cell of the invention is capable of using arabinose. A cell of theinvention is therefore, be capable of converting L-arabinose intoL-ribulose and/or xylulose 5-phosphate and/or into a desiredfermentation product, for example one of those mentioned herein.

Organisms, for example S. cerevisiae strains, able to produce ethanolfrom L-arabinose may be produced by modifying a cell introducing thearaA (L-arabinose isomerase), araB (L-ribulokinase) and araD(L-ribulose-5-P4-epimerase) genes from a suitable source. Such genes maybe introduced into a cell of the invention is order that it is capableof using arabinose. Such an approach is given is described inWO2003/095627. araA, araB and araD genes from Lactobacillus plantanummay be used and are disclosed in WO2008/041840. The araA gene fromBacillus subtilis and the araB and araD genes from Escherichia coli maybe used and are disclosed in EP1499708. In another embodiment, araA,araB and araD genes may derived from of at least one of the genusClavibacter, Arthrobacter and/or Gramella, in particular one ofClavibacter michiganensis, Arthrobacter aurescens, and/or Gramellaforsetii, as disclosed in WO 2009011591.

PPP-Genes

A cell of the invention may comprise one or more genetic modificationsthat increases the flux of the pentose phosphate pathway. In particular,the genetic modification(s) may lead to an increased flux through thenon-oxidative part pentose phosphate pathway. A genetic modificationthat causes an increased flux of the non-oxidative part of the pentosephosphate pathway is herein understood to mean a modification thatincreases the flux by at least a factor of about 1.1, about 1.2, about1.5, about 2, about 5, about 10 or about 20 as compared to the flux in astrain which is genetically identical except for the geneticmodification causing the increased flux. The flux of the non-oxidativepart of the pentose phosphate pathway may be measured by growing themodified host on xylose as sole carbon source, determining the specificxylose consumption rate and subtracting the specific xylitol productionrate from the specific xylose consumption rate, if any xylitol isproduced. However, the flux of the non-oxidative part of the pentosephosphate pathway is proportional with the growth rate on xylose as solecarbon source, preferably with the anaerobic growth rate on xylose assole carbon source. There is a linear relation between the growth rateon xylose as sole carbon source (μ_(max)) and the flux of thenon-oxidative part of the pentose phosphate pathway. The specific xyloseconsumption rate (Q_(s)) is equal to the growth rate (μ) divided by theyield of biomass on sugar (Y_(xs)) because the yield of biomass on sugaris constant (under a given set of conditions: anaerobic, growth medium,pH, genetic background of the strain, etc.; i.e. Q_(s)=μ/Y_(xs)).Therefore the increased flux of the non-oxidative part of the pentosephosphate pathway may be deduced from the increase in maximum growthrate under these conditions unless transport (uptake is limiting).

One or more genetic modifications that increase the flux of the pentosephosphate pathway may be introduced in the host cell in various ways.These including e.g. achieving higher steady state activity levels ofxylulose kinase and/or one or more of the enzymes of the non-oxidativepart pentose phosphate pathway and/or a reduced steady state level ofunspecific aldose reductase activity. These changes in steady stateactivity levels may be effected by selection of mutants (spontaneous orinduced by chemicals or radiation) and/or by recombinant DNA technologye.g. by overexpression or inactivation, respectively, of genes encodingthe enzymes or factors regulating these genes.

In a preferred host cell, the genetic modification comprisesoverexpression of at least one enzyme of the (non-oxidative part)pentose phosphate pathway. Preferably the enzyme is selected from thegroup consisting of the enzymes encoding for ribulose-5-phosphateisomerase, ribulose-5-phosphate epimerase, transketolase andtransaldolase. Various combinations of enzymes of the (non-oxidativepart) pentose phosphate pathway may be overexpressed. E.g. the enzymesthat are overexpressed may be at least the enzymes ribulose-5-phosphateisomerase and ribulose-5-phosphate epimerase; or at least the enzymesribulose-5-phosphate isomerase and transketolase; or at least theenzymes ribulose-5-phosphate isomerase and transaldolase; or at leastthe enzymes ribulose-5-phosphate epimerase and transketolase; or atleast the enzymes ribulose-5-phosphate epimerase and transaldolase; orat least the enzymes transketolase and transaldolase; or at least theenzymes ribulose-5-phosphate epimerase, transketolase and transaldolase;or at least the enzymes ribulose-5-phosphate isomerase, transketolaseand transaldolase; or at least the enzymes ribulose-5-phosphateisomerase, ribulose-5-phosphate epimerase, and transaldolase; or atleast the enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphateepimerase, and transketolase. In one embodiment of the invention each ofthe enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphateepimerase, transketolase and transaldolase are overexpressed in the hostcell. More preferred is a host cell in which the genetic modificationcomprises at least overexpression of both the enzymes transketolase andtransaldolase as such a host cell is already capable of anaerobic growthon xylose. In fact, under some conditions host cells overexpressing onlythe transketolase and the transaldolase already have the same anaerobicgrowth rate on xylose as do host cells that overexpress all four of theenzymes, i.e. the ribulose-5-phosphate isomerase, ribulose-5-phosphateepimerase, transketolase and transaldolase. Moreover, host cellsoverexpressing both of the enzymes ribulose-5-phosphate isomerase andribulose-5-phosphate epimerase are preferred over host cellsoverexpressing only the isomerase or only the epimerase asoverexpression of only one of these enzymes may produce metabolicimbalances.

The enzyme “ribulose 5-phosphate epimerase” (EC 5.1.3.1) is hereindefined as an enzyme that catalyses the epimerisation of D-xylulose5-phosphate into D-ribulose 5-phosphate and vice versa. The enzyme isalso known as phosphoribulose epimerase; erythrose-4-phosphateisomerase; phosphoketopentose 3-epimerase; xylulose phosphate3-epimerase; phosphoketopentose epimerase; ribulose 5-phosphate3-epimerase; D-ribulose phosphate-3-epimerase; D-ribulose 5-phosphateepimerase; D-ribulose-5-P 3-epimerase; D-xylulose-5-phosphate3-epimerase; pentose-5-phosphate 3-epimerase; or D-ribulose-5-phosphate3-epimerase. A ribulose 5-phosphate epimerase may be further defined byits amino acid sequence. Likewise a ribulose 5-phosphate epimerase maybe defined by a nucleotide sequence encoding the enzyme as well as by anucleotide sequence hybridising to a reference nucleotide sequenceencoding a ribulose 5-phosphate epimerase. The nucleotide sequenceencoding for ribulose 5-phosphate epimerase is herein designated RPE1.

The enzyme “ribulose 5-phosphate isomerase” (EC 5.3.1.6) is hereindefined as an enzyme that catalyses direct isomerisation of D-ribose5-phosphate into D-ribulose 5-phosphate and vice versa. The enzyme isalso known as phosphopentosisomerase; phosphoriboisomerase; ribosephosphate isomerase; 5-phosphoribose isomerase; D-ribose 5-phosphateisomerase; D-ribose-5-phosphate ketol-isomerase; or D-ribose-5-phosphatealdose-ketose-isomerase. A ribulose 5-phosphate isomerase may be furtherdefined by its amino acid sequence. Likewise a ribulose 5-phosphateisomerase may be defined by a nucleotide sequence encoding the enzyme aswell as by a nucleotide sequence hybridising to a reference nucleotidesequence encoding a ribulose 5-phosphate isomerase. The nucleotidesequence encoding for ribulose 5-phosphate isomerase is hereindesignated RKI1.

The enzyme “transketolase” (EC 2.2.1.1) is herein defined as an enzymethat catalyses the reaction: D-ribose 5-phosphate+D-xylulose5-phosphate<−>sedoheptulose 7-phosphate+D-glyceraldehyde 3-phosphate andvice versa. The enzyme is also known as glycolaldehydetransferase orsedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphateglycolaldehydetransferase. A transketolase may be further defined by itsamino acid. Likewise a transketolase may be defined by a nucleotidesequence encoding the enzyme as well as by a nucleotide sequencehybridising to a reference nucleotide sequence encoding a transketolase.The nucleotide sequence encoding for transketolase is herein designatedTKL1.

The enzyme “transaldolase” (EC 2.2.1.2) is herein defined as an enzymethat catalyses the reaction: sedoheptulose 7-phosphate+D-glyceraldehyde3-phosphate<−>D-erythrose 4-phosphate+D-fructose 6-phosphate and viceversa. The enzyme is also known as dihydroxyacetonetransferase;dihydroxyacetone synthase; formaldehyde transketolase; orsedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphateglyceronetransferase. A transaldolase may be further defined by itsamino acid sequence. Likewise a transaldolase may be defined by anucleotide sequence encoding the enzyme as well as by a nucleotidesequence hybridising to a reference nucleotide sequence encoding atransaldolase. The nucleotide sequence encoding for transketolase fromis herein designated TAL1.

Xylose Isomerase or Xylose Reductase and Xylitol Dehydrogenase Genes

According to the invention, one, two or more copies of one or morexylose isomerase gene and/or one or more xylose reductase and xylitoldehydrogenase are introduced into the genome of the host cell. Thepresence of these two or more genetic elements confers on the cell theability to convert xylose by isomerisation or reduction.

In one embodiment, the one, two or more copies of one or more xyloseisomerase gene are introduced into the genome of the host cell.

A “xylose isomerase” (EC 5.3.1.5) is herein defined as an enzyme thatcatalyses the direct isomerisation of D-xylose into D-xylulose and/orvice versa. The enzyme is also known as a D-xylose ketoisomerase. Axylose isomerase herein may also be capable of catalysing the conversionbetween D-glucose and D-fructose (and accordingly may therefore bereferred to as a glucose isomerase). A xylose isomerase herein mayrequire a bivalent cation, such as magnesium, manganese or cobalt as acofactor.

Accordingly, such a mixed sugar cell is capable of isomerising xylose toxylulose. The ability of isomerising xylose to xylulose is conferred onthe host cell by transformation of the host cell with a nucleic acidconstruct comprising a nucleotide sequence encoding a defined xyloseisomerase. A mixed sugar cell isomerises xylose into xylulose by thedirect isomerisation of xylose to xylulose.

A unit (U) of xylose isomerase activity may herein be defined as theamount of enzyme producing 1 nmol of xylulose per minute, underconditions as described by Kuyper et al. (2003, FEMS Yeast Res. 4:69-78). The Xylose isomerise gene may have various origin, such as forexample Pyromyces sp. as disclosed in WO2006/009434. Other suitableorigins are Bacteroides, in particular Bacteroides uniformis asdescribed in PCT/EP2009/52623, Bacillus, in particular Bacillusstearothermophilus as described in PCT/EP2009/052625.

In another embodiment, the two or more copies of one or more xylosereductase and xylitol dehydrogenase genes are introduced into the genomeof the host cell. In this embodiment the conversion of xylose isconducted in a two step conversion of xylose into xylulose via a xylitolintermediate as catalysed by xylose reductase and xylitol dehydrogenase,respectively. In an embodiment thereof xylose reductase (XR), xylitoldehydrogenase (XDH), and xylokinase (XK) may be overexpressed, andoptionally one or more of genes encoding NADPH producing enzymes areup-regulated and one or more of the genes encoding NADH consumingenzymes are up-regulated, as disclosed in WO 2004085627.

XKS1 Gene

A cell of the invention may comprise one or more genetic modificationsthat increase the specific xylulose kinase activity. Preferably thegenetic modification or modifications causes overexpression of axylulose kinase, e.g. by overexpression of a nucleotide sequenceencoding a xylulose kinase. The gene encoding the xylulose kinase may beendogenous to the host cell or may be a xylulose kinase that isheterologous to the host cell. A nucleotide sequence used foroverexpression of xylulose kinase in the host cell of the invention is anucleotide sequence encoding a polypeptide with xylulose kinaseactivity.

The enzyme “xylulose kinase” (EC 2.7.1.17) is herein defined as anenzyme that catalyses the reaction ATP+D-xylulose=ADP+D-xylulose5-phosphate. The enzyme is also known as a phosphorylating xylulokinase,D-xylulokinase or ATP: D-xylulose 5-phosphotransferase. A xylulosekinase of the invention may be further defined by its amino acidsequence. Likewise a xylulose kinase may be defined by a nucleotidesequence encoding the enzyme as well as by a nucleotide sequencehybridising to a reference nucleotide sequence encoding a xylulosekinase.

In a cell of the invention, a genetic modification or modifications thatincrease(s) the specific xylulose kinase activity may be combined withany of the modifications increasing the flux of the pentose phosphatepathway as described above. This is not, however, essential.

Thus, a host cell of the invention may comprise only a geneticmodification or modifications that increase the specific xylulose kinaseactivity. The various means available in the art for achieving andanalysing overexpression of a xylulose kinase in the host cells of theinvention are the same as described above for enzymes of the pentosephosphate pathway. Preferably in the host cells of the invention, axylulose kinase to be overexpressed is overexpressed by at least afactor of about 1.1, about 1.2, about 1.5, about 2, about 5, about 10 orabout 20 as compared to a strain which is genetically identical exceptfor the genetic modification(s) causing the overexpression. It is to beunderstood that these levels of overexpression may apply to the steadystate level of the enzyme's activity, the steady state level of theenzyme's protein as well as to the steady state level of the transcriptcoding for the enzyme.

Aldose Reductase (GRE3) Gene Deletion

In the embodiment, where XI is used as gene to convert xylose, it may beadvantageoud to reduce aldose reductase activity. A cell of theinvention may therefore comprise one or more genetic modifications thatreduce unspecific aldose reductase activity in the host cell.Preferably, unspecific aldose reductase activity is reduced in the hostcell by one or more genetic modifications that reduce the expression ofor inactivates a gene encoding an unspecific aldose reductase.Preferably, the genetic modification(s) reduce or inactivate theexpression of each endogenous copy of a gene encoding an unspecificaldose reductase in the host cell (herein called GRE3 deletion). Hostcells may comprise multiple copies of genes encoding unspecific aldosereductases as a result of di-, poly- or aneu-ploidy, and/or the hostcell may contain several different (iso)enzymes with aldose reductaseactivity that differ in amino acid sequence and that are each encoded bya different gene. Also in such instances preferably the expression ofeach gene that encodes an unspecific aldose reductase is reduced orinactivated. Preferably, the gene is inactivated by deletion of at leastpart of the gene or by disruption of the gene, whereby in this contextthe term gene also includes any non-coding sequence up- or down-streamof the coding sequence, the (partial) deletion or inactivation of whichresults in a reduction of expression of unspecific aldose reductaseactivity in the host cell.

A nucleotide sequence encoding an aldose reductase whose activity is tobe reduced in the host cell of the invention is a nucleotide sequenceencoding a polypeptide with aldose reductase activity.

Thus, a host cell of the invention comprising only a geneticmodification or modifications that reduce(s) unspecific aldose reductaseactivity in the host cell is specifically included in the invention.

The enzyme “aldose reductase” (EC 1.1.1.21) is herein defined as anyenzyme that is capable of reducing xylose or xylulose to xylitol. In thecontext of the present invention an aldose reductase may be anyunspecific aldose reductase that is native (endogenous) to a host cellof the invention and that is capable of reducing xylose or xylulose toxylitol. Unspecific aldose reductases catalyse the reaction:aldose+NAD(P)H+H⁺

alditol+NAD(P)⁺

The enzyme has a wide specificity and is also known as aldose reductase;polyol dehydrogenase (NADP⁺); alditol:NADP oxidoreductase; alditol:NADP⁺1-oxidoreductase; NADPH-aldopentose reductase; or NADPH-aldosereductase.

A particular example of such an unspecific aldose reductase that isendogenous to S. cerevisiae and that is encoded by the GRE3 gene (Traffet al., 2001, Appl. Environ. Microbiol. 67: 5668-74). Thus, an aldosereductase of the invention may be further defined by its amino acidsequence. Likewise an aldose reductase may be defined by the nucleotidesequences encoding the enzyme as well as by a nucleotide sequencehybridising to a reference nucleotide sequence encoding an aldosereductase.

Sequence Identity

Sequence identity (or sequence similarity) is herein defined as arelationship between two or more amino acid (polypeptide or protein)sequences or two or more nucleic acid (polynucleotide) sequences, asdetermined by comparing the sequences. Usually, sequence identities orsimilarities are compared, typically over the whole length of thesequences compared. However, sequences may be compared over shortercomparison windows. In the art, “identity” also means the degree ofsequence relatedness between amino acid or nucleic acid sequences, asthe case may be, as determined by the match between strings of suchsequences.

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include e.g. the BestFit, BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990),publicly available from NCBI and other sources (BLAST Manual, Altschul,S., et al., NCBI NLM NIH Bethesda, Md. 20894). Preferred parameters foramino acid sequences comparison using BLASTP are gap open 11.0, gapextend 1, Blosum 62 matrix. Preferred parameters for nucleic acidsequences comparison using BLASTP are gap open 11.0, gap extend 1, DNAfull matrix (DNA identity matrix).

Optionally, in determining the degree of amino acid similarity, theskilled person may also take into account so-called “conservative” aminoacid substitutions, as will be clear to the skilled person.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine.

Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine. Substitutional variants of theamino acid sequence disclosed herein are those in which at least oneresidue in the disclosed sequences has been removed and a differentresidue inserted in its place. Preferably, the amino acid change isconservative. Preferred conservative substitutions for each of thenaturally occurring amino acids are as follows: Ala to ser; Arg to lys;Asn to gln or his; Asp to glu; Cys to ser or ala; Gln to asn; Glu toasp; Gly to pro; His to asn or gln; He to leu or val; Leu to ile or val;Lys to arg; gln or glu; Met to leu or ile; Phe to met, leu or tyr; Serto thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile orleu.

Stringent hybridisation conditions are herein defined as conditions thatallow a nucleic acid sequence of at least about 25, preferably about 50nucleotides, 75 or 100 and most preferably of about 200 or morenucleotides, to hybridise at a temperature of about 65° C. in a solutioncomprising about 1 M salt, preferably 6×SSC (sodium chloride, sodiumcitrate) or any other solution having a comparable ionic strength, andwashing at 65° C. in a solution comprising about 0.1 M salt, or less,preferably 0.2×SSC or any other solution having a comparable ionicstrength. Preferably, the hybridisation is performed overnight, i.e. atleast for 10 hours and preferably washing is performed for at least onehour with at least two changes of the washing solution. These conditionswill usually allow the specific hybridisation of sequences having about90% or more sequence identity.

Moderate conditions are herein defined as conditions that allow anucleic acid sequences of at least 50 nucleotides, preferably of about200 or more nucleotides, to hybridise at a temperature of about 45° C.in a solution comprising about 1 M salt, preferably 6×SSC or any othersolution having a comparable ionic strength, and washing at roomtemperature in a solution comprising about 1 M salt, preferably 6×SSC orany other solution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours, andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having up to 50% sequence identity.The person skilled in the art will be able to modify these hybridisationconditions in order to specifically identify sequences varying inidentity between 50% and 90%.

To increase the likelihood that the introduced enzyme is expressed inactive form in a cell of the invention, the corresponding encodingnucleotide sequence may be adapted to optimise its codon usage to thatof the chosen yeast cell. Several methods for codon optimisation areknown in the art. A preferred method to optimise codon usage of thenucleotide sequences to that of the yeast is a codon pair optimizationtechnology as disclosed in WO2006/077258 and/or WO2008/000632.WO2008/000632 addresses codon-pair optimization. Codon-pair optimisationis a method wherein the nucleotide sequences encoding a polypeptide aremodified with respect to their codon-usage, in particular thecodon-pairs that are used, to obtain improved expression of thenucleotide sequence encoding the polypeptide and/or improved productionof the encoded polypeptide. Codon pairs are defined as a set of twosubsequent triplets (codons) in a coding sequence.

As a simple measure for gene expression and translation efficiency,herein, the Codon Adaptation Index (CAI), as described in Xuhua Xia,Evolutionary Bioinformatics 2007: 3 53-58, is used. The index uses areference set of highly expressed genes from a species to assess therelative merits of each codon, and a score for a gene is calculated fromthe frequency of use of all codons in that gene. The index assesses theextent to which selection has been effective in moulding the pattern ofcodon usage. In that respect it is useful for predicting the level ofexpression of a gene, for assessing the adaptation of viral genes totheir hosts, and for making comparisons of codon usage in differentorganisms. The index may also give an approximate indication of thelikely success of heterologous gene expression. In the codon pairoptimized genes according to the invention, the CAI is 0.6 or more, 0.7or more, 0.8 or more, 0.85 or more, 0.87 or more 0.90 or more, 0.95 ormore, or about 1.0.

A cell of the invention is thus a cell that comprises, i.e. has beentransformed with, a nucleic acid construct comprising the nucleotidesequence encoding the araA, araB and araD genes as defined above. Thenucleic acid construct comprising araA coding sequence preferably iscapable of expression of the araA genes in the host cell.

Preferably, the genes are expressed in the cytosol. Cytosolic expressionmay be achieved by deletion or modification of a mitochondrial orperoxisomal targeting signal.

Bioproducts Production

Over the years suggestions have been made for the introduction ofvarious organisms for the production of bio-ethanol from crop sugars. Inpractice, however, all major bio-ethanol production processes havecontinued to use the yeasts of the genus Saccharomyces as ethanolproducer. This is due to the many attractive features of Saccharomycesspecies for industrial processes, i.e., a high acid-, ethanol- andosmo-tolerance, capability of anaerobic growth, and of course its highalcoholic fermentative capacity. Preferred yeast species as host cellsinclude S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum,S. diastaticus, K. lactis, K. marxianus or K. fragilis.

A cell of the invention may be able to convert plant biomass,celluloses, hemicelluloses, pectins, rhamnose, galactose, frucose,maltose, maltodextrines, ribose, ribulose, or starch, starchderivatives, sucrose, lactose and glycerol, for example into fermentablesugars. Accordingly, a cell of the invention may express one or moreenzymes such as a cellulase (an endocellulase or an exocellulase), ahemicellulase (an endo- or exo-xylanase or arabinase) necessary for theconversion of cellulose into glucose monomers and hemicellulose intoxylose and arabinose monomers, a pectinase able to convert pectins intoglucuronic acid and galacturonic acid or an amylase to convert starchinto glucose monomers.

The cell further preferably comprises those enzymatic activitiesrequired for conversion of pyruvate to a desired fermentation product,such as ethanol, butanol, lactic acid, 3-hydroxy-propionic acid, acrylicacid, acetic acid, succinic acid, citric acid, fumaric acid, malic acid,itaconic acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, aβ-lactam antibiotic or a cephalosporin.

A preferred cell of the invention is a cell that is naturally capable ofalcoholic fermentation, preferably, anaerobic alcoholic fermentation. Acell of the invention preferably has a high tolerance to ethanol, a hightolerance to low pH (i.e. capable of growth at a pH lower than about 5,about 4, about 3, or about 2.5) and towards organic acids like lacticacid, acetic acid or formic acid and/or sugar degradation products suchas furfural and hydroxy-methylfurfural and/or a high tolerance toelevated temperatures.

Any of the above characteristics or activities of a cell of theinvention may be naturally present in the cell or may be introduced ormodified by genetic modification.

A cell of the invention may be a cell suitable for the production ofethanol. A cell of the invention may, however, be suitable for theproduction of fermentation products other than ethanol. Suchnon-ethanolic fermentation products include in principle any bulk orfine chemical that is producible by a eukaryotic microorganism such as ayeast or a filamentous fungus.

Such fermentation products may be, for example, butanol, lactic acid,3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, itaconic acid, an amino acid,1,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic or acephalosporin. A preferred cell of the invention for production ofnon-ethanolic fermentation products is a host cell that contains agenetic modification that results in decreased alcohol dehydrogenaseactivity.

In a further aspect the invention relates to fermentation processes inwhich the cells of the invention are used for the fermentation of acarbon source comprising a source of xylose, such as xylose. In additionto a source of xylose the carbon source in the fermentation medium mayalso comprise a source of glucose. The source of xylose or glucose maybe xylose or glucose as such or may be any carbohydrate oligo- orpolymer comprising xylose or glucose units, such as e.g. lignocellulose,xylans, cellulose, starch and the like. For release of xylose or glucoseunits from such carbohydrates, appropriate carbohydrases (such asxylanases, glucanases, amylases and the like) may be added to thefermentation medium or may be produced by the cell. In the latter casethe cell may be genetically engineered to produce and excrete suchcarbohydrases. An additional advantage of using oligo- or polymericsources of glucose is that it enables to maintain a low(er)concentration of free glucose during the fermentation, e.g. by usingrate-limiting amounts of the carbohydrases. This, in turn, will preventrepression of systems required for metabolism and transport ofnon-glucose sugars such as xylose.

In a preferred process the cell ferments both the xylose and glucose,preferably simultaneously in which case preferably a cell is used whichis insensitive to glucose repression to prevent diauxic growth. Inaddition to a source of xylose (and glucose) as carbon source, thefermentation medium will further comprise the appropriate ingredientrequired for growth of the cell. Compositions of fermentation media forgrowth of microorganisms such as yeasts are well known in the art. Thefermentation process is a process for the production of a fermentationproduct such as e.g. ethanol, butanol, lactic acid, 3-hydroxy-propionicacid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid,fumaric acid, itaconic acid, an amino acid, 1,3-propane-diol, ethylene,glycerol, a β-lactam antibiotic, such as Penicillin G or Penicillin Vand fermentative derivatives thereof, and a cephalosporin.

Lignocellulose

Lignocellulose, which may be considered as a potential renewablefeedstock, generally comprises the polysaccharides cellulose (glucans)and hemicelluloses (xylans, heteroxylans and xyloglucans). In addition,some hemicellulose may be present as glucomannans, for example inwood-derived feedstocks. The enzymatic hydrolysis of thesepolysaccharides to soluble sugars, including both monomers andmultimers, for example glucose, cellobiose, xylose, arabinose,galactose, fructose, mannose, rhamnose, ribose, galacturonic acid,glucoronic acid and other hexoses and pentoses occurs under the actionof different enzymes acting in concert.

In addition, pectins and other pectic substances such as arabinans maymake up considerably proportion of the dry mass of typically cell wallsfrom non-woody plant tissues (about a quarter to half of dry mass may bepectins).

Pretreatment

Pretreatment may be desirable to release sugars that may be fermentedaccording to the invention from the lignocellulosic (includinghemicellulosic) material. This steps may be executed with conventionalmethods, e.g.

Enzymatic Hydrolysis

Enzymatic hydrolysis may be executed with conventional methods.

Fermentation

The fermentation process may be an aerobic or an anaerobic fermentationprocess. An anaerobic fermentation process is herein defined as afermentation process run in the absence of oxygen or in whichsubstantially no oxygen is consumed, preferably less than about 5, about2.5 or about 1 mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e.oxygen consumption is not detectable), and wherein organic moleculesserve as both electron donor and electron acceptors. In the absence ofoxygen, NADH produced in glycolysis and biomass formation, cannot beoxidised by oxidative phosphorylation. To solve this problem manymicroorganisms use pyruvate or one of its derivatives as an electron andhydrogen acceptor thereby regenerating NAD⁺.

Thus, in a preferred anaerobic fermentation process pyruvate is used asan electron (and hydrogen acceptor) and is reduced to fermentationproducts such as ethanol, butanol, lactic acid, 3-hydroxy-propionicacid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid,fumaric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, aβ-lactam antibiotic and a cephalosporin.

The fermentation process is preferably run at a temperature that isoptimal for the cell. Thus, for most yeasts or fungal host cells, thefermentation process is performed at a temperature which is less thanabout 42° C., preferably less than about 38° C. For yeast or filamentousfungal host cells, the fermentation process is preferably performed at atemperature which is lower than about 35, about 33, about 30 or about28° C. and at a temperature which is higher than about 20, about 22, orabout 25° C.

The ethanol yield on xylose and/or glucose in the process preferably isat least about 50, about 60, about 70, about 80, about 90, about 95 orabout 98%. The ethanol yield is herein defined as a percentage of thetheoretical maximum yield.

The invention also relates to a process for producing a fermentationproduct.,

The fermentation processes may be carried out in batch, fed-batch orcontinuous mode. A separate hydrolysis and fermentation (SHF) process ora simultaneous saccharification and fermentation (SSF) process may alsobe applied. A combination of these fermentation process modes may alsobe possible for optimal productivity.

The fermentation process according to the present invention may be rununder aerobic and anaerobic conditions. Preferably, the process iscarried out under micro-aerophilic or oxygen limited conditions.

An anaerobic fermentation process is herein defined as a fermentationprocess run in the absence of oxygen or in which substantially no oxygenis consumed, preferably less than about 5, about 2.5 or about 1mmol/L/h, and wherein organic molecules serve as both electron donor andelectron acceptors.

An oxygen-limited fermentation process is a process in which the oxygenconsumption is limited by the oxygen transfer from the gas to theliquid. The degree of oxygen limitation is determined by the amount andcomposition of the ingoing gasflow as well as the actual mixing/masstransfer properties of the fermentation equipment used. Preferably, in aprocess under oxygen-limited conditions, the rate of oxygen consumptionis at least about 5.5, more preferably at least about 6, such as atleast 7 mmol/L/h. A process of the invention comprises recovery of thefermentation product.

In a preferred process the cell ferments both the xylose and glucose,preferably simultaneously in which case preferably a cell is used whichis insensitive to glucose repression to prevent diauxic growth. Inaddition to a source of xylose (and glucose) as carbon source, thefermentation medium will further comprise the appropriate ingredientrequired for growth of the cell. Compositions of fermentation media forgrowth of microorganisms such as yeasts are well known in the art

The fermentation processes may be carried out in batch, fed-batch orcontinuous mode. A separate hydrolysis and fermentation (SHF) process ora simultaneous saccharification and fermentation (SSF) process may alsobe applied. A combination of these fermentation process modes may alsobe possible for optimal productivity. These processes are describedhereafter in more detail.

SSF Mode

For Simultaneous Saccharification and Fermentation (SSF) mode, thereaction time for liquefaction/hydrolysis or presaccharification step isdependent on the time to realize a desired yield, i.e. cellulose toglucose conversion yield. Such yield is preferably as high as possible,preferably 60% or more, 65% or more, 70% or more, 75% or more 80% ormore, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more,98% or more, 99% or more, even 99.5% or more or 99.9% or more.

According to the invention very high sugar concentrations in SHF modeand very high product concentrations (e.g. ethanol) in SSF mode arerealized. In SHF operation the glucose concentration is 25 g/L or more,30 g/L or more, 35 g/L or more, 40 g/L or more, 45 g/L or more, 50 g/Lor more, 55 g/L or more, 60 g/L or more, 65 g/L or more, 70 g/L or more,75 g/L or more, 80 g/L or more, 85 g/L or more, 90 g/L or more, 95 g/Lor more, 100 g/L or more, 110 g/L or more, 120 g/L or more or may e.g.be 25 g/L-250 g/L, 30 g1/L-200 g/L, 40 g/L-200 g/L, 50 g/L-200 g/L, 60g/L-200 g/L, 70 g/L-200 g/L, 80 g/L-200 g/L, 90 g/L, 80 g/L-200 g/L.

Product Concentration in SSF Mode

In SSF operation, the product concentration (g/L) is dependent on theamount of glucose produced, but this is not visible since sugars areconverted to product in the SSF, and product concentrations can berelated to underlying glucose concentration by multiplication with thetheoretical mamimum yield (Yps max in gr product per gram glucose)

The theoretical maximum yield (Yps max in gr product per gram glucose)of a fermentation product can be derived from textbook biochemistry. Forethanol, 1 mole of glucose (180 gr) yields according to normalglycolysis fermentation pathway in yeast 2 moles of ethanol (=2×46=92 grethanol. The theoretical maximum yield of ethanol on glucose istherefore 92/180=0.511 gr ethanol/gr glucose.

For Butanol (MW 74 gr/mole) or iso butanol, the theoretical maximumyield is 1 mole of butanol per mole of glucose. So Yps max for(iso-)butanol=74/180=0.411 gr (iso-)butanol/gr glucose.

For lactic acid the fermentation yield for homolactic fermentation is 2moles of lactic acid (MW=90 gr/mole) per mole of glucose. According tothis stoichiometry, the Yps max=1 gr lactic acid/gr glucose.

For other fermentation products a similar calculation may be made.

SSF Mode

In SSF operation the product concentration is 25 g*Yps g/L/L or more,30*Yps g/L or more, 35 g*Yps/L or more, 40*Yps g/L or more, 45*Yps g/Lor more, 50*Yps g/L or more, 55*Yps g/L or more, 60*Yps g/L or more,65*Yps g/L or more, 70*Yps g/L or more, 75*Yps g/L or more, 80*Yps g/Lor more, 85*Yps g/L or more, 90*Yps g/L or more, 95*Yps g/L or more,100*Yps g/L or more, 110*Yps g/L or more, 120 g/L*Yps or more or maye.g. be 25*Yps g/L-250*Yps g/L, 30*Yps gl/L-200*Yps g/L, 40*Ypsg/L-200*Yps g/L, 50*Yps g/L-200*Yps g/L, 60*Yps g/L-200*Yps g/L, 70*Ypsg/L-200*Yps g/L, 80*Yps g/L-200*Yps g/L, 90*Yps g/L, 80*Yps g/L-200*Ypsg/L

Accordingly, the invention provides a method for the preparation of afermentation product, which method comprises:

a. degrading lignocellulose using a method as described herein; and

b. fermenting the resulting material,

thereby to prepare a fermentation product.

Fermentation Product

The fermentation product of the invention may be any useful product. Inone embodiment, it is a product selected from the group consisting ofethanol, n-butanol, isobutanol, lactic acid, 3-hydroxy-propionic acid,acrylic acid, acetic acid, succinic acid, fumaric acid, malic acid,itaconic acid, maleic acid, citric acid, adipic acid, an amino acid,such as lysine, methionine, tryptophan, threonine, and aspartic acid,1,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic and acephalosporin, vitamins, pharmaceuticals, animal feed supplements,specialty chemicals, chemical feedstocks, plastics, solvents, fuels,including biofuels and biogas or organic polymers, and an industrialenzyme, such as a protease, a cellulase, an amylase, a glucanase, alactase, a lipase, a lyase, an oxidoreductases, a transferase or axylanase.

Recovery of the Fermentation Product

For the recovery of the fermenation product existing technologies areused. For different fermentation products different recovery processesare appropriate. Existing methods of recovering ethanol from aqueousmixtures commonly use fractionation and adsorption techniques. Forexample, a beer still can be used to process a fermented product, whichcontains ethanol in an aqueous mixture, to produce an enrichedethanol-containing mixture that is then subjected to fractionation(e.g., fractional distillation or other like techniques). Next, thefractions containing the highest concentrations of ethanol can be passedthrough an adsorber to remove most, if not all, of the remaining waterfrom the ethanol.

The following examples illustrate the invention:

EXAMPLES

Unless indicated otherwise, the methods used are standard biochemicaltechniques. Examples of suitable general methodology textbooks includeSambrook et al., Molecular Cloning, a Laboratory Manual (1989) andAusubel et al., Current Protocols in Molecular Biology (1995), JohnWiley & Sons, Inc.

Transformation of S. cerevisiae

Transformation of S. cerevisiae was done as described by Gietz and Woods(2002; Transformation of the yeast by the LiAc/SS carrier DNA/PEGmethod. Methods in Enzymology 350: 87-96).

Colony PCR

A single colony isolate was picked with a plastic toothpick andresuspended in 50 μl milliQ water. The sample was incubated for 10minutes at 99° C. 5 μl of the incubated sample was used as a templatefor the PCR reaction, using Phusion® DNA polymerase (Finnzymes)according to the instructions provided by the supplier.

PCR reaction conditions:

step 1   3′ 98° C. step 2 10″ 98° C. step 3 15″ 58° C. repeat step 2 to4 for 30 cycles step 4 30″ 72° C. step 5   4′ 72° C. step 6 30″ 20° C.

Medium Composition

Growth experiments: Saccharomyces cerevisiae strains are grown on mediumhaving the following composition: 0.67% (w/v) yeast nitrogen base orsynthetic medium (Verduyn et al., Yeast 8:501-517, 1992) and eitherglucose, arabinose, galactose or xylose, or a combination of thesesubstrates (see below). For agar plates the medium is supplemented with2% (w/v) bacteriological agar.

Ethanol production: cultivations were performed at 30° C. in 100 mlsynthetic model medium (Verduyn-medium (Verduyn et al., Yeast 8:501-517,1992) with 5% glucose, 5% xylose, 3.5% arabinose and 1-1.5% galactose)in the BAM (Biological Activity Monitor, Halotec, The Netherlands). ThepH of the medium was adjusted to 4.2 with 2 M NaOH/H2SO4 prior tosterilisation. The synthetic medium for anaerobic cultivation wassupplemented with 0.01 g I-1 ergosterol and 0.42 g I-1 Tween 80dissolved in ethanol (Andreasen and Stier. J. Cell Physiol. 41:23-36,1953; and Andreasen and Stier. J. Cell Physiol. 43:271-281, 1954).Cultures were stirred by magnetic stirrer. Anaerobic conditionsdeveloped rapidly during fermentation as the culture was not aerated.CO2 production was monitored constantly. Sugar conversion and productformation was analyzed by NMR. Growth was monitored by following opticaldensity of the culture at 600 nm on a LKB Ultrospec K spectrophotometer.

Pre-cultures were prepared by inoculating 25 ml Verduyn-medium (Verduynet al., Yeast 8:501-517, 1992) supplemented with 2% glucose in a 100-mlshake flask with a frozen stock culture or a single colony from agarplate. After incubation at 30° C. in an orbital shaker (200 rpm) forapproximately 24 hours, this culture was harvested and used forinoculation of the BAM at an OD 600 of approximately 2.

Example 1 Introduction of the Genes araA, araB and araD into the Genomeof S. cerevisiae

1.1 Construction of an Expression Vector Containing the Genes forArabinose Pathway

Plasmid pPWT018, as set out in FIG. 2, was constructed as follows:vector pPWT006 (FIG. 1, consisting of a SIT2-locus (Gottlin-Ninfa andKaback (1986) Molecular and Cell Biology vol. 6, no. 6, 2185-2197) andthe markers allowing for selection of transformants on the antibioticG418 and the ability to grow on acetamide (vide supra), was digestedwith the restriction enzymes BsiWI and MluI. The kanMX-marker,conferring resistance to G418, was isolated from p427TEF (DualsystemsBiotech) and a fragment containing the amdS-marker has been described inthe literature (Swinkels, B. W., Noordermeer, A. C. M. and Renniers, A.C. H. M (1995) The use of the amdS cDNA of Aspergillus nidulans as adominant, bidirectional selectable marker for yeast transformation.Yeast Volume 11, Issue 1995A, page S579; and U.S. Pat. No. 6,051,431).The genes encoding arabinose isomerase (araA), L-ribulokinase (araB) andL-ribulose-5-phosphate-4-epimerase (araD) from Lactobacillus plantarum,as disclosed in patent application WO2008/041840, were synthesized byBaseClear (Leiden, The Netherlands). One large fragment was synthesized,harbouring the three arabinose-genes mentioned above, under control of(or operable linked to) strong promoters from S. cerevisiae, i.e. theTDH3-promoter controlling the expression of the araA-gene, theENO1-promoter controlling the araB-gene and the PGI1-promotercontrolling the araD-gene. This fragment was surrounded by the uniquerestriction enzymes Acc65I and MluI. Cloning of this fragment intopPWT006 digested with MluI and BsiWI, resulted in plasmid pPWT018 (FIG.2). The sequence of plasmid pPWT018 is set out in SEQ ID 17.

1.2 Yeast Transformation

CEN.PK113-7D (MATa URA3 HIS3 LEU2 TRP1 MAL2-8 SUC2) was transformed withplasmid pPWT018, which was previously linearized with SfiI (New EnglandBiolabs), according to the instructions of the supplier. A syntheticSfiI-site was designed in the 5′-flank of the SIT2-gene (see FIG. 2).Transformation mixtures were plated on YPD-agar (per liter: 10 grams ofyeast extract, 20 grams per liter peptone, 20 grams per liter dextrose,20 grams of agar) containing 100 μg G418 (Sigma Aldrich) per ml. Aftertwo to four days, colonies appeared on the plates, whereas the negativecontrol (i.e. no addition of DNA in the transformation experiment)resulted in blank YPD/G418-plates. The integration of plasmid pPWT018 isdirected to the SIT2-locus. Transformants were characterized using PCRand Southern blotting techniques.

PCR reactions, which are indicative for the correct integration of onecopy of plasmid pPWT018, were performed with the primers indicated bySEQ ID 18 and 15, and 15 and 14 (see FIG. 4). With the primer pairs ofSEQ ID 18 and 15, the correct integration at the SIT2-locus was checked.If plasmid pPWT018 was integrated in multiple copies (head-to-tailintegration), the primer pair of SEQ ID 15 and 14 will give aPCR-product. If the latter PCR product is absent, this is indicative forone copy integration of pPWT018. A strain in which one copy of plasmidpPWT018 was integrated in the SIT2-locus was designated BIE104R2.

1.3 Marker Rescue

In order to be able to transform the yeast strain with other constructs,using the same selection markers, it is necessary to remove theselectable markers. The design of plasmid pPWT018 was such, that uponintegration of pPWT018 in the chromosome, homologous sequences are inclose proximity of each other. This design allows the selectable markersto be lost by spontaneous intramolecular recombination of thesehomologous regions.

Upon vegetative growth, intramolecular recombination will take place,although at low frequency. The frequency of this recombination dependson the length of the homology and the locus in the genome (unpublishedresults). Upon sequential transfer of a subfraction of the culture tofresh medium, intramolecular recombinants will accumulate in time.

To this end, strain BIE104R2 was cultured in YPD-medium (per liter: 10grams of yeast extract, 20 grams per liter peptone, 20 grams per literdextrose), starting from a single colony isolate. 25 μl of an overnightculture was used to inoculate fresh YPD medium. After at least five ofsuch serial transfers, the optical density of the culture was determinedand cells were diluted to a concentration of approximately 5000 per ml.100 μl of the cell suspension was plated on Yeast Carbon Base medium(Difco) containing 30 mM KPi (pH 6.8), 0.1% (NH4)2SO4, 40 mMfluoro-acetamide (Amersham) and 1.8% agar (Difco). Cells identical tocells of strain BIE104R2, i.e. without intracellular recombination,still contain the amdS-gene. To those cells, fluoro-acetamide is toxic.These cells will not be able to grow and will not form colonies on amedium containing fluoro-acetamide. However, if intramolecularrecombination has occurred, BIE104R2-variants that have lost theselectable markers will be able to grow on the fluoro-acetamide medium,since they are unable to convert fluoro-acetamide into growth inhibitingcompounds. Those cells will form colonies on this agar medium.

The thus obtained fluoro-acetamide resistant colonies were subjected toPCR analysis using primers of SEQ ID 18 and 15, and 14 and 19. Primersof SEQ ID 18 and 5 will give a band if recombination of the selectablemarkers has taken place as intended. As a result, the cassette with thegenes araA, araB and araD under control of the strong yeast promotershave been integrated in the SIT2-locus of the genome of the host strain.In that case, a PCR reaction using primers of SEQ ID 14 and 19 shouldnot result in a PCR product, since primer 14 primes in a region thatshould be lost due to recombination. If a band is obtained with thelatter primers, this is indicative for the presence of the completeplasmid pPWT018 in the genome, so no recombination has taken place.

If primers of SEQ ID 18 and 15 do not result in a PCR product,recombination has taken place, but in such a way that the completeplasmid pPWT018 has recombined out of the genome. Not only were theselectable markers lost, but also the arabinose-genes. In fact,wild-type yeast has been retrieved.

Isolates that showed PCR results in accordance with one copy integrationof pPWT018 were subjected to Southern blot analysis. The chromosomal DNAof strains CEN.PK113-7D and the correct recombinants were digested withEcoRI and HindIII (double digestion). A SIT2-probe was prepared withprimers of SEQ ID 20 and 21, using chromosomal DNA of CEN.PK113-7D as atemplate. The result of the hybridisation experiment is shown in FIG. 3.The expected hybridisation pattern may be deduced from the physical mapsas set out in FIG. 4 (panels a and b).

In the wild-type strain, a band of 2.35 kb is observed, which is inaccordance with the expected size of the wild type gene (FIG. 4, panela). Upon integration and partial loss by recombination of the plasmidpPWT018, a band of 1.06 kb was expected (FIG. 4, panel b). Indeed, thisband is observed, as shown in FIG. 3 (lane 2).

One of the strains that showed the correct pattern of bands on theSouthern blot (as can be deduced from FIG. 3) is the strain designatedas BIE104A2.

1.4 Introduction of Four Constitutively Expressed Genes of theNon-Oxidative Pentose Phosphate Pathway

Saccharomyces cerevisiae BIE104A2, expressing the genes araA, araB andaraD constitutively, was transformed with plasmid pPWT080 (FIG. 5). Thesequence of plasmid pPWT080 is set out in SEQ ID NO: 4. The procedurefor transformation and selection, after selecting a one copytransformant, are the same as described above in sections 1.1, 1.2 and1.3). In short, BIE104A2 was transformed with SfiI-digested pPWT080.Transformation mixtures were plated on YPD-agar (per liter: 10 grams ofyeast extract, 20 grams per liter peptone, 20 grams per liter dextrose,20 grams of agar) containing 100 μg G418 (Sigma Aldrich) per ml.

After two to four days, colonies appeared on the plates, whereas thenegative control (i.e. no addition of DNA in the transformationexperiment) resulted in blank YPD/G418-plates.

The integration of plasmid pPWT080 is directed to the GRE3-locus.Transformants were characterized using PCR and Southern blottingtechniques.

A transformant showing correct integration of one copy of plasmidpPWT080, in accordance with the expected hybridisation pattern, wasdesignated BIE104A2F1.

In order to be able to introduce the genes encoding xylose isomerase andxylulokinase (example 5), it is necessary to remove the selectionmarkers introduced by the integration of plasmid pPWT080. To this end,strain BIE104A2F1 was cultured in YPD-medium, starting from a colonyisolate. 25 μl of an overnight culture was used to inoculate freshYPD-medium. After five serial transfers, the optical density of theculture was determined and cells were diluted to a concentration ofapproximately 5000 per ml. 100 μl of the cell suspension was plated onYeast Carbon Base medium (Difco) containing 30 mM KPi (pH 6.8), 0.1%(NH4)2SO4, 40 mM fluoro-acetamide (Amersham) and 1.8% agar (Difco).Fluoro-acetamide resistant colonies were subjected to PCR analysis and,in case of correct PCR-profiles, Southern blot analysis (section 1.3 ofexample 1). One of the strains that showed the correct pattern of bandson the Southern blot is the strain designated as BIE104A2P1.

Example 2 Adaptive Evolution

2.1 Adaptive Evolution (Aerobically)

Single colony isolate of strain BIE104A2P1 was used to inoculateYNB-medium (Difco) supplemented with 2% galactose. The preculture wasincubated for approximately 24 hours at 30° C. and 280 rpm. Cells wereharvested and inoculated in YNB medium containing 1% galactose and 1%arabinose at a starting OD600 of 0.2 (FIG. 8). Cells were grown at 30°C. and 280 rpm. The optical density at 600 nm was monitored regularly.

When the optical density reached a value of 5, an aliquot of the culturewas transferred to fresh YNB medium containing the same medium. Theamount of cells added was such that the starting OD600 of the culturewas 0.2. After reaching an OD 600 of 5 again, an aliquot of the culturewas transferred to YNB medium containing 2% arabinose as sole carbonsource (event indicated by (1) in FIG. 8).

Upon transfer to YNB with 2% arabinose as sole carbon source growthcould be observed after approximately two weeks. When the opticaldensity at 600 nm reached a value at least of 1, cells were transferredto a shake flask with fresh YNB-medium supplemented with 2% arabinose ata starting OD600 of 0.2 (FIG. 8).

Sequential transfer was repeated three times, as is set it in FIG. 8.The resulting strain which was able to grow fast on arabinose wasdesignated BIE104A2P1c.

2.2 Adaptive Evolution (Anaerobically)

After adaptation on growth on arabinose under aerobic conditions, asingle colony from strain BIE104A2P1c was inoculated in YNB mediumsupplemented with 2% glucose. The preculture was incubated forapproximately 24 hours at 30° C. and 280 rpm. Cells were harvested andinoculated in YNB medium containing 2% arabinose, with optical densityOD⁶⁰⁰ of 0.2. The flasks were closed with waterlocks, ensuring anaerobicgrowth conditions after the oxygen was exhausted from the medium andhead space. After reaching an OD 600 minimum of 3, an aliquot of theculture was transferred to fresh YNB medium containing 2% arabinose(FIG. 9), each with optical density OD⁶⁰⁰ of 0.2.

After several transfers the resulting strain was designated BIE104A2P1d(=BIE201).

Example 3 Fermentative Capacity Determination

Single colony isolates of strain BIE104, BIE104A2P1c and BIE201 wereused to inoculate YNB-medium (Difco) supplemented with 2% glucose. Theprecultures were incubated for approximately 24 hours at 30° C. and 280rpm. Cells were harvested and inoculated in a synthetic model medium(Verduyn et al., Yeast 8:501-517, 1992; 5% glucose, 5% xylose, 3.5%arabinose, 1% galactose) at an initial OD600 of approximately 2 in theBAM. CO2 production was monitored constantly. Sugar conversion andproduct formation was analyzed by NMR. The data represent the residualamount of sugars at the indicated (glucose, arabinose, galactose andxylose in grams per liter) and the formation of (by-)products (ethanol,glycerol). Growth was monitored by following optical density of theculture at 600 nm (FIG. 10, 11, 12). The experiment was running forapproximately 140 hours.

The experiments clearly show that reference strain BIE104 convertedglucose rapidly, but was not able to convert neither arabinose norgalactose within 140 hours (FIG. 10). However, strain BIE104A2P1c andBIE201 were capable to convert arabinose and galactose (FIGS. 11 and 12,respectively). Galactose and arabinose utilization started immediatelyafter glucose depletion after less than 20 hours. Both sugars wereconverted simultaneously. However, strain BIE201 which was improved forarabinose growth under anaerobic conditions, consumed both sugars morerapidly (FIG. 12). In all fermentations only glycerol was generated asby-product. The data of the fermentation of BIE201 are given herein intable 2.

TABLE 2 Sugar concentrations and ethanol concentrations (g/l) of BIE201fermenta- tion as shown in FIG. 12. Maximal ethanol concentration iscalculated by multiplying concentrations by 0.51 for each sugar andsummarizing. Eth- anol concentration at 136 h (39.2 g/l) means anethanol yield of 0.45 eth- anol/g sugar. This yield shows that allsugars were converted to ethanol. Concentrations in g/l Time (h) Glu XylAra Gal EtOH 0 42.8 50.2 31.6 12.9 0.7 16 0.1 54.2 35.8 10.8 22.9 23 0.049.2 31.3 8.4 18.7 39 0.1 52.8 16.3 0.7 32.1 48 0.0 52.5 8.9 0.2 29.4 650.0 55.1 4.3 0.3 40.3 111 0.0 48.8 0.5 0.3 38.1 136 0.0 49.6 0.2 0.339.2 Maximal ethanol concentrations (in g/l) from Glucose 21.8 Arabinose16.1 Galactose 6.6 Total 44.5 Experimental ethanol yield 0.45 gethanol/g sugar

From this calculation it is clear that the sugars glucose, galactose andarabinose are each converted into ethanol.

Example 4 Effect of the PPP-Genes on Sugar Conversion

To test the effect of the PPP-genes on sugar conversion, single coloniesfrom strain BIE104A2 and BIE105A2 were inoculated in YNB-medium (Difco)supplemented with 2% glucose. Both strains contain the arabinose-genesand were evolved for growth on arabinose (as described in example 2,section 2.1). Strain BIE105A2 has the background of an industrialstrain. However, it was transformed with the same methods and constructsas described before (example 1, section 1.2).

Precultures were harvested and inoculated in synthetic corn fiber modelmedium (Verduyn et al., Yeast 8:501-517, 1992; 5% glucose, 5% xylose,3.5% arabinose, 1.5% galactose) with a starting OD 600 of approximately2 in the BAM. CO2 production was monitored constantly. Sugar conversionand product formation was analyzed by NMR. The data represent theresidual amount of sugars at the indicated (glucose, arabinose,galactose and xylose in grams per liter) and the formation of(by-)products (ethanol, glycerol). Growth was monitored by followingoptical density of the culture at 600 nm. The experiment was running forapproximately 160 hours.

The experiments show that both strains are capable to convert arabinoseand galactose immediately after glucose depletion without theoverexpression of the PPP-genes (FIGS. 13 and 14).

Example 5 Introduction of Constitutively Expressed Genes Encoding XyloseIsomerase and Xylulokinase

5.1 Yeast Transformation

Strain BIE104A2P1 (MATa URA3 HIS3 LEU2 TRP1 MAL2-8 SUC2SIT2::[TDH3-araA, ENO1-araB, PGI1-araD] ΔGRE3::[TPI1p-TAL1, ADH1p-TKL1,PGI1p-RPE1, ENO1p-RKI1]) was transformed with plasmid pPWT042 (FIG. 16).Plasmid pPWT042 derives from vector pPWT007 (FIG. 15). It contains thecodon pair optimized xylulokinase from S. cerevisiae and the codon-pairoptimized xylose isomerase from Bacteroides uniformis (SEQ 2) asdisclosed in patent application PCT/EP2009/52623. Prior to thetransformation of BIE104A2P1, pPWT042 was linearized using therestriction enzyme SfiI, according to the instructions provided by thesupplier. Transformation mixtures were plated on YPD-agar (per liter: 10grams of yeast extract, 20 grams per liter peptone, 20 grams per literdextrose, 20 grams of agar) containing 100 μg G418 (Sigma Aldrich) perml.

After two to four days, colonies appeared on the plates, whereas thenegative control (i.e. no addition of DNA in the transformationexperiment) resulted in blank YPD/G418-plates.

Upon digestion of plasmid pPWT042 with SfiI, its integration is directedto the SIT4-locus (Gottlin-Ninfa and Kaback (1986) Molecular andCellular Biology Vol. 6, No. 6, 2185-2197) in the genome (FIG. 17).Transformants were characterized using PCR and Southernblottingtechniques, as described in example 1 (section 1.2).

A strain with one copy of plasmid pPWT042 integrated into the genome wasdesignated BIE104A2P1Y9.

5.2 Growth Experiments

Single colony isolates of strains BIE104A2P1Y9 were used to inoculateYNB-medium (Difco) supplemented with 2% glucose or 2% galactose. Theinoculated flasks were incubated at 30° C. and 280 rpm until the opticaldensity at 600 nm reached a value of at least 2.0.

YNB-medium supplemented with 1% arabinose and 1% xylose was inoculatedwith the overnight cultures at a starting OD600 of 0.2. Cells were grownat 30° C. and 280 rpm. The optical density at 600 nm was monitoredregularly. When the optical density reached a value larger than 2.0, analiquot of the culture was transferred to fresh YNB medium containing 2%xylose and 0.2% arabinose. The amount of cells added was such that thestarting OD600 of the culture was 0.2.

The optical density was monitored regularly. The results are shown inFIG. 18, panel a (precultures on galactose) and panel b (precultures onglucose).

The results clearly show that the strains are capable of utilizingglucose, galactose, arabinose and xylose.

5.3 Marker Rescue

To remove the selection marker introduced by the integration of plasmidpPWT042, the strain BIE104A2P1Y9 was cultured in YPD-medium, startingfrom a colony isolate. 25 μl of an overnight culture was used toinoculate fresh YPD-medium. After serial transfers, the optical densityof the culture was determined and cells were diluted to a concentrationof approximately 5000 per ml. 100 μl of the cell suspension was platedon Yeast Carbon Base medium (Difco) containing 30 mM KPi (pH 6.8), 0.1%(NH4)2SO4, 40 mM fluoro-acetamide (Amersham) and 1.8% agar (Difco).Fluoro-acetamide resistant colonies were subjected to PCR analysis and,in case of correct PCR-profiles, Southern blot analysis (section 1.3,example 1). One of the strains that showed the correct pattern of bandson the Southern blot is the strain designated as BIE104A2P1X9.

5.4 Growth Experiments

Single colony isolates of strain BIE104A2P1X9 (BIE104A2P1×9a1 andBIE104A2P1×9a2) were used to inoculate Verduyn-medium (Difco)supplemented with 2% glucose. The inoculated flasks were incubated at30° C. and 280 rpm for approximately 24 hours.

Verduyn-medium supplemented with 2% xylose was inoculated with theovernight cultures at a starting OD600 of 0.2. Cells were grown at 30°C. and 280 rpm. The optical density at 600 nm was monitored regularly.The results are shown in FIG. 19.

The results clearly show that both independent colonies of strainBIE104A2P1X9 are still capable to utilize xylose after marker rescue. Aswas already shown in example 3, the strain is capable to utilizeglucose, arabinose and galactose (FIG. 11 and FIG. 12).

Example 6 Transformation of S. cerevisiae for Succinic Acid Productionon Arabinose and Galactose

6.1. Expression Constructs

Expression construct pGBS414PPK-3 comprising a phosphoenol pyruvatecarboxykinase PCKa (E.C. 4.1.1.49) from Actinobacillus succinogenes, andglycosomal fumarate reductase FRDg (E.C. 1.3.1.6) from Trypanosomabrucei, and an expression construct pGBS415FUM3 comprising a fumarase(E.C. 4.2.1.2.) from Rhizopus oryzae, and a peroxisomal malatedehydrogenase MDH3 (E.C. 1.1.1.37) are made as described previously inWO2009/065778 on p. 19-20, and 22-30 which herein enclosed by referenceincluding the figures and sequence listing.

Expression construct pGBS416ARAABD comprising the genes araA, araB andaraD, derived from Lactobacillus plantarum, are constructed by cloning aPCR product, comprising the araABD expression cassette from plasmidpPWT018, into plasmid pRS416. The PCR fragment is generated usingPhusion® DNA polymerase (Finnzymes) and PCR primers defined in here asSEQ ID 22 and SEQ ID 23. The PCR product is cut with the restrictionenzymes SalI and NotI, as is plasmid pRS416. After ligation andtransformation of E. coli TOP10, the right recombinants are selected onbasis of restriction enzyme analysis. The physical map of plasmidpGBS416ARAABD is set out in FIG. 20

6.2. S. cerevisiae Strains

The plasmids pGBS414PPK-3, pGBS415-FUM-3 are transformed into S.cerevisiae strain CEN.PK113-6B (MATA ura3-52 leu2-112 trp1-289). Inaddition plasmid pGBS416ARAABD is transformed into this yeast to createprototrophic yeast strains. The expression vectors are transformed intoyeast by electroporation. The transformation mixtures are plated onYeast Nitrogen Base (YNB) w/o AA (Difco)+2% glucose.

Strains are subjected to adapted evolution (see section 2) for growth onarabinose as sole carbon source.

6.3. Growth Experiments and Succinic Acid Production

Transformants are inoculated in 20 ml pre-culture medium consisting ofVerduyn medium (Verduyn et al., 1992, Yeast. July; 8(7):501-17)comprising 2% galactose (w/v) and grown under aerobic conditions in 100ml shake flasks in a shaking incubator at 30° C. at 250 rpm. Afterapproximately 24 hours, cells are transferred to fresh Verduyn mediumcontaining either 2% glucose, 2% galactose or 2% arabinose, or mixturesthereof, in fourfold. Two flasks are incubated under aerobic conditions,two flasks are incubated under anaerobic conditions, for instance byclosing the flasks using a waterlock or by incubation in an anaerobicorbital shaker. At time intervals, culture samples are taken. Thesamples are centrifuged for 5 minutes at 4750 rpm. 1 ml supernatant isused to measure succinic acid levels by HPLC as described in section6.4.

6.4. HPLC Analysis

HPLC is performed for the determination of organic acids and sugars in.The principle of the separation on a Phenomenex Rezex-RHM-Monosaccharidecolumn is based on size exclusion, ion-exclusion and ion-exchange usingreversed phase mechanisms. Detection takes place by differentialrefractive index and ultra violet detectors.

LITERATURE

Lit. No Source (1) Bioresource Technology 1994 Vol. 47 page 283-284 (2)Micard, Enzyme Microbiol Technology 1996 Vol 19 page 163-170 (3) DOERadke, Idaho wheat straw composition (4) Grohman and Botast ProcessBiochemistry 1997 Vol. 32 No 5 405-415 (5) Saska B&B 1995 517-523 (6)PCT/EP2009/52623 (7) Zheng Appl. Biochem. Microbiol. 2007, Vol. 136-140pp 423-436 (8) Bradshaw Appl Biochem. Microbiol. 2007 Vol 136-140 page395-406 (9) Cara Appl Biochem. Microbiol. 2007 Vol 136-140 page 379-394

The invention claimed is:
 1. A process for the production of at leastone fermentation product from a sugar composition comprising glucose,galactose and arabinose, said process comprising: a) fermenting saidsugar composition in the presence of a yeast recombinant belonging tothe genera Saccharomyces, Kluyveromyces, Candida, Pichia,Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces and/orYarrowia, and b) recovering the fermentation product, wherein saidrecombinant yeast comprises gene araA, araB and araD, wherein each ofsaid glucose, galactose and arabinose is converted into at least onefermentation product, and wherein said at least one fermentation productis selected from the group consisting of: ethanol, n-butanol,isobutanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, aceticacid, succinic acid, fumaric acid, malic acid, itaconic acid, maleicacid, citric acid, adipic acid, an amino acid, 1,3-propane-diol,ethylene, glycerol, a β-lactam antibiotic, a cephalosporin, andvitamins.
 2. The process according to claim 1, wherein said fermentationproduct comprises ethanol.
 3. The process according to claim 1, whereinsaid sugar composition is produced from lignocellulosic material by: a)pretreatment of at least one lignocellulosic material to producepretreated lignocellulosic material; b) enzymatic treatment of thepretreated lignocellulosic material to produce said sugar composition.4. The process according to claim 1, wherein said yeast belongs to thegenus Saccharomyces.
 5. The process according to claim 4, wherein saidyeast is a-Saccharomyces cerevisiae.
 6. The process according to claim1, wherein said yeast comprises a deletion of an aldose reductase gene.7. The process according to claim 6, wherein said yeast comprisesxylA-gene and/or XKS1-gene.
 8. The process according to claim 7, whereinsaid genes are introduced in said yeast by introduction into a host cella nucleic acid construct comprising: a) a cluster comprising PPP geneTAL1, TKL1, RPE1 and/or RKI1, under control of strong promoters, b) acluster comprising a xylA-gene and/or XKS1-gene under control ofconstitutive promoter, c) a cluster comprising gene araA, araB and araDand/or a cluster of xylA-gene and/or XKS1-gene; and d) deletion of analdose reductase gene; and adaptive evolution of said yeast.
 9. Theprocess according to claim 8, wherein said host cell is an inhibitortolerant cell.
 10. The process according to claim 8, wherein said hostcell is an industrial strain.
 11. The process according to claim 1,wherein fermentation is conducted under anaerobic and/or oxygen limitedconditions.
 12. The process according to claim 1, wherein said aminoacid is selected from the group consisting of lysine, methionine,tryptophan, threonine, and aspartic acid.